Binding of a neutralizing antibody to dengue virus alters the

Transcription

© 2008 Nature Publishing Group http://www.nature.com/nsmb
ARTICLES
Binding of a neutralizing antibody to dengue virus alters
the arrangement of surface glycoproteins
Shee-Mei Lok1, Victor Kostyuchenko1, Grant E Nybakken2, Heather A Holdaway1, Anthony J Battisti1,
Soila Sukupolvi-Petty3, Dagmar Sedlak1,6, Daved H Fremont2, Paul R Chipman1, John T Roehrig4,
Michael S Diamond2,3,5, Richard J Kuhn1 & Michael G Rossmann1
The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by
inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the
viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue
virus. Nevertheless, antibody could bind to the virus at 37 1C, suggesting that the virus is in dynamic motion making hidden
epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the
organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric
units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.
Dengue, yellow fever and West Nile viruses are major human pathogens that are members of the flavivirus genus of the Flaviviridae1
family. Dengue virus (DENV) is the causative agent for dengue fever
and the more severe dengue hemorrhagic fever (DHF). Although
DENV infects approximately 50–100 million people each year2, no
effective vaccine has been licensed for human use. Vaccine development has been hampered by the potential complications following
secondary DENV infections, which can result in DHF3,4. This frequently occurs when the secondary infection is of a serotype different
from the first, possibly because cross-reactive non-neutralizing antibodies from the first infection bind to the virus and promote
antibody-dependent enhancement (ADE) of infection in cells expressing Fc-g receptors. To avoid ADE, a tetravalent vaccine would need
to elicit strongly neutralizing antibodies against all four serotypes
of DENV.
Flaviviruses consist of an icosahedrally symmetric ectodomain,
containing 180 copies of the envelope (E) glycoprotein and 180 copies
of the membrane (M) protein anchored in and surrounding a lipid
membrane5. The nucleocapsid core within the membrane consists of a
positive-sense, 11-kb RNA genome and multiple copies of the capsid
protein. In the mature virus, the E protein is arranged into 30 rafts of
three parallel dimers5. Crystal structures of the dimeric E protein6–8
have been determined for DENV serotypes 2 and 3. The E-protein
monomer has three domains, E-DI, E-DII and E-DIII, of which E-DIII
is probably involved in recognition of the principal cell receptor9–12.
However, E-DII can also participate in the initial binding to an
ancillary receptor, DC-SIGN, thereby possibly enhancing the local
concentration of the primary receptor in the vicinity of the virus
required for cell entry13.
Neutralizing epitopes are clustered at the tip of E-DII (which is also
the location of the fusion peptide9,14), the hinge region between E-DI
and E-DII, and the lateral surface of E-DIII7,15. The hinge regions,
between E-DI and E-DII and between E-DI and E-DIII, participate in
structural rearrangements that occur at low pH as the immature virus
is converted into infectious particles8 and in the initial stages of
infection when the virus fuses with an endosomal membrane16,17.
Before the fusion of the virus to endosomal membranes, the
E dimers dissociate and then reassociate as trimers18. This conformational change can be blocked in West Nile virus (WNV) infection by
the monoclonal antibody (mAb) E16 binding to E-DIII19,20. The
postfusion, trimeric E-protein structures of tick-borne encephalitis
virus16 and DENV17 show that E-DIII rotates by about 701 closer to
E-DI relative to its position in the dimeric prefusion structure.
The mAb 1A1D-2 strongly neutralizes DENV serotypes 1, 2 and 3
(Supplementary Fig. 1 online) but does not bind to serotype 4
(ref. 21). We report here the crystal structure of the Fab fragment of
the mAb 1A1D-2 complexed with recombinant E-DIII of DENV
serotype 2. Interpretation of a cryo-electron microscopy (cryoEM)
image reconstruction of Fab 1A1D-2 complexed with DENV, made
by using this crystal structure, showed that Fab 1A1D-2 bound to only
1Department of Biological Sciences, Purdue University, 915 West State Street, West Lafayette, Indiana 47907-2054, USA. 2Department of Pathology & Immunology
and 3Department of Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA. 4Arbovirus Disease Branch,
Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services,
P.O. Box 2087, Fort Collins, Colorado 80522, USA. 5Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue,
St. Louis, Missouri 63110, USA. 6Present address: Solid State Chemistry, An Aptuit Company, 3065 Kent Avenue, West Lafayette, Indiana 47906, USA.
Correspondence should be addressed to M.G.R. (mr@purdue.edu).
Received 24 September 2007; accepted 4 January 2008; published online 10 February 2008; doi:10.1038/nsmb.1382
NATURE STRUCTURAL & MOLECULAR BIOLOGY
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1
ARTICLES
a
b
c
d
two of the three E proteins in each of the 60 icosahedral asymmetric
units and resulted in the rearrangement of E proteins, thus preventing
viral infection.
RESULTS
Crystal structure of the Fab–1A1D-2:E-DIII complex
The recombinant E-DIII protein of DENV serotype 2 was complexed
with Fab 1A1D-2. The structures were determined for two crystal
forms of the complex (Table 1 and Supplementary Fig. 2a online).
Both forms had one complex per crystallographic asymmetric unit.
Crystal form 1 diffracted to only 3.8-Å resolution, whereas form 2
diffracted to 3.0-Å resolution. The r.m.s. deviation between equivalent
Ca atoms of the two structures using only the constant domain of Fab,
the variable domain of Fab or E-DIII were 1.2 Å, 1.7 Å and 1.2 Å,
respectively. In addition, the E-DIII structure in the complex was
similar to the E-DIII structure in crystallized DENV E protein6,8,
with an r.m.s. difference of only 1.1 Å between equivalent Ca atoms.
a
Figure 1 Interactions of E-DIII with the Fab
1A1D-2 molecule and comparison of Fab 1A1D-2
binding to DENV E-DIII with Fab E16 binding to
WNV (Protein Data Bank accession code
1ZTW)20. (a) Stereo diagram of the interface
between E-DIII and Fab 1A1D-2 in the crystal
structure complex. Residues of Fab 1A1D-2 and
E-DIII are colored yellow and cyan, respectively.
The light (L) and heavy (H) chain of the Fab
molecule are indicated in the residue label with
subscripts. Oxygen and nitrogen atoms are
colored red and blue, respectively. Putative
hydrogen bonds are shown as dotted lines.
(b) Footprints of Fab 1A1D-2 (cyan) and E16
(pink) on E-DIII. Residues recognized by both
Fabs are colored purple. (c) Ribbon diagram of
the E molecule onto which the E-DIII components
of the Fab 1A1D-2 and E16 complex crystal
structures have been superimposed. E-DI, E-DII
and E-DIII are colored red, yellow and blue,
respectively. The Fab 1A1D-2 (cyan) and Fab
E16 (pink) molecules are shown as Ca
backbones. (d) The pseudo atomic–resolution
structure of the E-protein arrangement in mature
dengue virus (DENV)31. The E-protein molecules
at the A, B and C sites in the asymmetric unit of
the virus are shaded.
The main difference between the two crystal forms is a 201 change in
the elbow angle of the Fab molecule (Supplementary Fig. 2b),
consistent with previous observations that the hinge angle in Fab
fragments is flexible19,22.
The surface area of the interface between the Fab molecule and
E-DIII is 940 Å2, typical of antibody-antigen interactions23. The
binding surface on the Fab molecule consists of five of the six
complementary determining regions L1, L2, H1, H2 and H3. The
E-DIII binding surface is predominantly on one b-strand (residues
305–312), but residues 325, 364, 388 and 390 also contact the Fab
molecule (Fig. 1a and Supplementary Table 1a online). There are
likely to be about eight hydrogen bonds, three salt bridges and some
hydrophobic interactions in the interface. The relative importance of
the residues in the contact area was examined by investigating whether
site-specific mutant forms of E-DIII (Supplementary Fig. 3 online)
could bind to mAb 1A1D-2 using a yeast surface-display system.
Mutations of K305E, K307E or K310E completely abolished antibody
b
c
Figure 2 Fab 1A1D-2:DENV complex formation is temperature dependent. (a) DENV with no antibody. (b) DENV incubated with Fab 1A1D-2 at room
temperature showed few particles with bound Fab molecules. (c) DENV incubated with Fab 1A1D-2 at 37 1C showed that most particles were saturated with
Fab molecules.
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ARTICLES
Figure 3 CryoEM structure of the Fab 1A1D-2:DENV complex. (a) Stereo
diagram of the cryoEM reconstruction of DENV complexed with Fab 1A1D-2
at 24-Å resolution. Surface features that are greater than 240 Å
(corresponding to the external radius of the uncomplexed mature virus) from
the center of the virus are colored yellow, and the remaining surface is
colored red. The black triangle represents the asymmetric unit of the virus.
(b) Stereo diagram of an asymmetric unit of the cryoEM structure showing
the E proteins at the A and C sites complexed with Fab 1A1D-2 molecules
and the uncomplexed E molecule at site B. The E molecules with bound
antibody at sites A and C have their fusion peptides pointing toward the viral
membrane. The Fab 1A1D-2 molecule, E-DI, E-DII, E-DIII and fusion tip are
colored cyan, red, yellow, blue and green, respectively.
a
b
bound by Fab 1A1D-2 when incubated for 30 min at room temperature, whereas nearly all the particles bound Fab at 37 1C. This
suggested that higher temperature promotes increased mobility of
the E proteins on the surface of the virus, thus transiently exposing the
previously hidden part of the epitope and making it available for
antibody binding.
binding24, consistent with the crystal structure, which showed that
these residues participate in making salt bridges and hydrogen bonds
with the Fab molecule.
The importance of the b-strand, formed by residues 305–312
(b-strand A9), to the binding of various other neutralizing antibodies
against DENV is evident in that several escape mutations to neutralizing antibodies are located within the strand25,26. This region is also
recognized by the antibody 4E11, which cross-reacts with and neutralizes all DENV serotypes27,28.
Comparison of the amino acids in the 1A1D-2 DENV2 epitope
with other serotypes (Supplementary Table 1b calculated using the
program ClustalW, http://www.ebi.ac.uk/Tools/clustalw/), indicated
that the DENV2 epitope is closely similar to DENV1 and DENV3,
but not DENV4 (similarity score of 45, 45 and 18, respectively). This is
consistent with the inability of the antibody to bind DENV4 (ref. 21).
Comparison of mAb 1A1D-2 and E16 binding sites on E-DIII
The neutralizing mAb E16 inhibits WNV infection at a step after
attachment, but before fusion20, whereas the mAb 1A1D-2 neutralizes
DENV infection of Vero cells, in part by preventing viral attachment10.
Although both antibodies bind E-DIII, their footprints barely overlap
one another (Fig. 1b,c). Presumably, this difference, at least in part, is
likely to account for the difference in their mechanisms of neutralization. The surface accessibility of the E16 and 1A1D-2 epitopes on
E-DIII, as determined by crystallography, are different for each of the
three E molecules in the native virus icosahedral asymmetric unit,
identified as A, B and C in Figure 1d. The epitope recognized by E16
has an area of 1,550 Å2 and is fully exposed on E-DIIIB (E-DIII at
B site) and E-DIIIC (E-DIII at C site) of the mature virus, whereas
only 54% of the epitope is exposed for the potential binding site on
E-DIIIA (E-DIII at A site). This is consistent with the cryoEM
reconstruction map of Fab E16 complexed with WNV19, which
shows Fab molecules binding only to E-DIIIB and E-DIIIC, but
not to E-DIIIA.
In contrast to Fab E16, 18% of the binding surface of Fab 1A1D-2 is
buried at all three E-DIIIs in the icosahedral asymmetric unit of the
mature virus. This raises the question of how the antibody could bind
to any of the E-DIII epitopes. Temperature was found to be crucial
in the binding of the antibody (Fig. 2). Inspection of cryoEM
micrographs showed that fewer than one-third of the particles were
Structure of dengue virus 2 complexed with Fab 1A1D-2
The cryoEM reconstruction of Fab 1A1D-2 complexed with DENV2
incubated at 37 1C was determined to 24-Å resolution (Fig. 3).
Interpretation of the cryoEM map showed that the E glycoproteins
on the viral surface had undergone a major rearrangement (Fig. 4).
Such large quaternary structural changes are not uncommon in
flaviviruses, as occurs in the maturation of virions29 and in the fusion
of virions to cell membranes17,18.
The interpretation of the cryoEM map (Fig. 3a) was initially guided
by the two large, flat features in each icosahedral asymmetric unit,
presumably representing the Fab molecules. Therefore, the first fitting
operation was to manually position the 1A1D-2:E-DIII crystal structure into the appropriate densities. This showed that the elbow angle
found in crystal 2 (Supplementary Fig. 2b) correlated better with the
cryoEM density at both of these sites. The initial manual fitting results
were optimized using the EMfit program30 (Supplementary Fig. 4
and Supplementary Table 2b online). The positions of all E-DIII
domains in the antibody complex were similar to those in the
native virus (Figs. 1d and 3b). Location of E-DI-DII at sites A and
C (Fig. 3b) in the cryoEM map were predicted by superimposing
E-DIII of the E protein as found in the mature virus31 onto the
already positioned E-DIIIs complex structure. The E-DI-DII at sites A
and C then had to be translated about 15 Å and 8 Å, respectively,
to satisfy the nearest densities. The remaining uninterpreted
density corresponded to the E molecule at site B (Supplementary
Table 2b).
The resultant structure had the parallel E molecules at sites A, B and
C in the mature virus (Fig. 1d) rotated counterclockwise (viewed from
outside the virus) by roughly 331, 321 and 731, respectively (Fig. 3b).
As a result, the epitopes on E-DIIIA and E-DIIIC had become
completely exposed and their associated E-DIIs had their fusion tip
pointing toward the viral lipid envelope (Fig. 3b and Supplementary
Fig. 5a–c online). The E molecules at site C seem to make dimers by
pairing across the icosahedral two-fold axes (Supplementary Fig. 5b).
The E protein at site B had its Fab binding epitope turned inwards.
Comparison of the E-protein structures at the A, B and C sites with
the structure of the E protein in the mature virus showed that the
unliganded E molecule corresponding to the B site had changed little
compared to its structure in the mature virus. However, the structure
of the other two E molecules with bound Fab molecules at sites A and
C caused a change in the position of E-DIII relative to E-DI-DII
(Supplementary Fig. 5d).
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ARTICLES
Figure 4 The E-protein organization undergoes major rearrangement. The
figure shows only the Ca chains of E proteins of the uncomplexed mature
dengue virus (DENV; left), and the Fab complex structure (right). The black
triangle represents the asymmetric unit of the virus. E-DI, E-DII and E-DIII
are colored red, yellow and blue, respectively.
DISCUSSION
The ability to expose the partially hidden epitope at all E-DIII sites can
be explained if it is assumed that icosahedral viruses can engage in
‘breathing’ motions. Dynamic structural changes have been demonstrated for icosahedral nodavirus32 and rhinovirus33. The breathing
exposes internal regions of rhinovirus structural proteins (VP1, VP2
and VP3), making them available for protease cleavage. The motion
can be inhibited by certain antiviral compounds that bind into a cavity
within VP1 (ref. 33), thereby stabilizing the virus and reducing the
extent of the breathing. Apparently, in the present case, the breathing
of DENV can expose the 18% of the hidden 1A1D-2 epitope for Fab
attachment when incubated at an elevated temperature (Fig. 2), but is
insufficient to expose 46% of the hidden surface at the WNV E-DIIIA
site to allow binding of Fab E16 (ref. 19). The ability of Fab 1A1D-2 to
bind to epitopes near the five-fold (the E-DIIIA site) and three-fold
(the E-DIIIC site) vertices, but not elsewhere on the viral surface,
suggests that the extent of the breathing motion might be greatest at
these vertices.
Although, presumably, breathing exposes the epitopes required for
1A1D-2 binding to DENV, breathing also implies that, in the absence
of antibody, the E proteins return to their original positions in the
mature virus. Once a Fab molecule has bound, the virus would
probably not be able to return to its previous range of dynamic
motions because of steric hindrance between the bound Fab molecules
and neighboring E proteins. Thus, the Fab molecules may capture
transient viral intermediate conformations. However, there is a problem with this concept. If the observed structure were in the normal
dynamic range of the viral conformations, then E16 should have
captured the same state, as its epitope on E-DIII near the five-fold
vertices is completely exposed in the cryoEM structure of the 1A1D-2:
virus complex. However, E16 Fabs did not bind to these sites, even at
37 1C (B. Kaufmann, Purdue University, unpublished data). Thus,
possibly, once a 1A1D-2 Fab molecule has bound to a transient
intermediate conformation, the E proteins plus bound Fab molecules
move to positions that do not occur during the normal breathing of
the native virus to relieve the steric stress. One caveat is that this
analysis assumes that DENV and WNV have the same breathing
motion, because E16 binds to WNV and not to DENV. Another
possibility is that, once E16 has bound to the fully available B and C
sites, then breathing might be diminished or altered, making binding
of E16 to any other normally partially hidden epitope impossible.
Thus, the structure of the 1A1D-2:virus complex might be a trapped
form of a breathing mode rather than a dead-end conformation.
4
If it is assumed that the particle will tend to conserve its icosahedral
symmetry during breathing, then as soon as one antibody has bound,
the rest of the virus alters its structure to the antibody-bound
conformation. Thus, the binding of one or only a few 1A1D-2
mAbs will make all other E proteins more accessible for antibody
binding. Hence, a low concentration of antibody would be sufficient
to alter the surface structure of the virus. This cascade mechanism is
supported by the observation that some particles seen on micrographs
of DENV incubated with Fab 1A1D-2 at room temperature were
covered by Fab molecules, whereas others had none (Fig. 2).
The mAb 1A1D-2 probably inhibits DENV attachment by several
different mechanisms. One block to infection could be the result
of the antibody altering the spatial distances between the glycans on
the E proteins and, therefore, inhibiting the interaction of the
virus with its ancillary attachment receptor, DC-SIGN13,34. Another
block to infection may be that the antibody binding to DIII of
the E glycoproteins prevents binding of the virus to its primary
entry receptor9,12.
An understanding of the mechanism of neutralization of an antibody could elucidate its likelihood for promoting ADE at a given
concentration. All neutralizing antibodies can promote ADE in vitro at
subneutralizing concentrations35–38. Recent stoichiometric analysis of
flavivirus antibody binding to virus suggests that there is a threshold
occupancy required for neutralization38. Concentrations of antibody
that fall below this threshold or antibodies that never reach this
threshold, even at maximal binding, will enhance infection in vitro in
cells expressing Fc-g receptors. The mechanism of antibody neutralization may determine the occupancy requirement for inhibition of
infection and, therefore, influence the range of concentration that
promotes ADE. Antibodies that neutralize solely by sterically blocking
receptor attachment may require higher concentrations for complete
neutralization and, therefore, have a greater range of concentration for
sustaining ADE. Indeed, this was recently observed with DII-specific
mAbs against WNV14. In contrast, mAbs such as 1A1D-2 that
promote a cascade of E-protein rearrangements on DENV may have
lower occupancy requirements for neutralization. Vaccines that consist
of an epitope recognized by mAb 1A1D-2 may thus elicit an antibody
response with greater inhibitory activity and less possibility of enhancing infection over a wider range of antibody concentration.
METHODS
Neutralization assay. To determine the concentration of mAb required to
reduce the number of plaques on BHK-21 cells, increasing concentrations of
mAb 1A1D-2 were added to 50 plaque-forming units (p.f.u.) of DENV and
incubated at 37 1C for 1 h. We then added 300 ml of the mixture to a monolayer
of BHK-21 cells in a 6-well plate and incubated for 1 h at 37 1C. Supernatant
was removed and 3 ml of 1.0% (w/v) carboxyl methyl cellulose in MEM plus
5% (v/v) FCS was layered onto the infected cells. After further incubation at
34 1C for 8 d, the wells were stained with 0.5% (w/v) crystal violet dissolved in
25% (v/v) formaldehyde to visualize the plaques. Percentage of plaque reduction was calculated as:
Percent plaque reduction ¼ 100 "
#!
"
$
plaque number when incubated with mAb
#100
plaque number without mAb
1A1D-2 neutralized DENV serotypes 1, 2 and 3, although DENV3 was least
sensitive to this antibody (Supplementary Fig. 1).
Crystallization of the 1A1D-2 Fab:E-DIII complex. A two-fold molar excess
of E-DIII (Supplementary Methods online) was added to 1A1D-2 Fab
(Supplementary Methods) and incubated at room temperature for 2 h. The
complex was purified with a Superdex 75 column (GE Healthcare) in 20 mM
HEPES, pH 7.5, 150 mM NaCl and 0.01% (w/v) sodium azide, and concentrated to 2 mg ml–1. Successful crystallization conditions were 0.1 M MES
ARTICLES
Table 1 Data collection, processing and refinement statistics
Crystal form 1
Crystal form 2
C2
C2
164.78, 66.89, 79.41
90, 113.1, 90
168.12, 58.65, 78.20
90, 114.4, 90
Resolution (Å)
Rmerge
3.8
0.118 (0.198)
3.0
0.107 (0.28)
I / sI
Completeness (%)
6 (3)
74.35 (54.3)
11 (3.5)
98.5 (90.1)
Redundancy
2.3 (1.7)
3.4 (2.8)
Refinement
Resolution (Å)
20–3.8
20–3.0
No. reflections
Rwork / Rfree
5,817 (509)
31/36
13,829 (1,553)
26/32
3,888
0
3,980
254
Data collection
Space group
Cell dimensions
a, b, c (Å)
a, b, g (1)
No. atoms
Protein
Water
B-factors
Protein
Water
r.m.s. deviations
Bond lengths (Å)
Bond angles (1)
20.0a
39
20.0a
34
0.01
2.69
0.01
2.27
Values in parentheses are for highest-resolution shell.
aB-factor
value is set to 20 and not refined because of low-resolution data.
pH 5.8, 12% (w/v) PEG 3350 and 0.1 M MES, pH 5.4 and 11–13% (w/v) PEG
2000. Both kinds of crystals were needle-shaped with maximum dimensions of
100 # 30 # 30 mm. The crystals were frozen using 25–28% (w/v) PEG 400 as a
cryoprotectant in addition to the mother liquor.
Crystallographic structure determination. Diffraction data of both crystal
forms were collected using a wavelength of 0.979 Å at 100 K on beamline 23ID
at the Advanced Photon Source. The data were indexed and scaled using
HKL2000 (ref. 39; Table 1). Both crystal forms belong to space group C2,
differing primarily in only a 10% change of the monoclinic b axis. The
structures of both crystal forms were solved by molecular replacement using
the program MOLREP40. Several immunoglobulin structures were used as
search models before a good solution was attained for each of the crystal forms
(PDB code 1A3R and 1BBD for crystal forms 1 and 2, respectively). The trial
structures were initially refined as rigid bodies using the CNS program
system41. Electron density corresponding to E-DIII was found to be adjacent
to the hypervariable region, as expected. The E-DIII and Fab 1A1D-2 molecules
(Supplementary Methods) were modeled into the electron density using the
program O42. Coordinates were refined with the program CNS41 using
reflections between 20-Å and 3.8-Å spacing for crystal form 1 and between
20-Å and 3.0-Å spacing for crystal form 2 (Table 1).
Mapping of mAb 1AID-2 epitope by yeast surface display. We expressed
DENV2 (strain 16681) E-DIII (residues 296–415) of E in yeast15 by engineering
BamHI and XhoI restriction enzyme sites at the 5¢ and 3¢ ends of the E-DIII
gene, using PCR amplification from an infectious cDNA clone43. This fragment
was cloned into the BamHI and XhoI sites of the pYD1 vector (Invitrogen) and
expressed in the Saccharomyces cerevisiae strain EBY100. Single point mutations
at residues Thr303, Lys305, Lys307, Lys310 and Asn390 were made using the
QuikChange mutagenesis kit (Stratagene). Yeast that expressed mutant serotype
2 DENV E proteins was incubated with 50 ml of mAb (25 mg ml–1) on ice for
30 min. The yeast was washed three times with PBS supplemented with
1 mg ml–1 BSA, incubated with a 1:500 dilution of a goat anti-mouse IgG
that had been conjugated to Alexa Fluor 647 (Invitrogen), and analyzed using a
Becton Dickinson FACSCaliber flow cytometer.
Cryo-electron microscopy image reconstruction. DENV2 New Guinea
C strain was mixed with Fab 1A1D-2 at a concentration of one molecule of
Fab for every E protein on the surface of the virus. The complex was incubated
for 30 min either at room temperature or at 37 1C, followed by 2 h at 4 1C.
Nearly all the particles incubated at 37 1C retained their ‘spikey-rough’
appearance after the temperature had been lowered to 4 1C, showing that
the antibody was still bound. In contrast, only about one-third of the particles
had a spikey-rough appearance when incubated at room temperature and then
kept at 4 1C. Thus, the presence of antibody complexed with the virus at 37 1C
on most of the particles showed that only a few or no particles had reverted to
their original conformation when the temperature was lowered back to 4 1C.
Preincubation at 4 1C before freezing might have improved the homogeneity of
the sample by reducing the ‘breathing’ motion.
The virus:FAb complex suspension was flash frozen on holey carbon grids in
liquid ethane. Micrographs of the frozen complex were made with a CM200
FEG transmission electron microscope (Philips) using a calibrated magnification of 51,040 and an electron dose of approximately 25 e– per Å2. The
micrographs were digitized with a Zeiss SCAI scanner with a 2.74-Å separation
between pixels. Particles were boxed and normalized using the program
EMAN44. The micrographs were underfocused by 2.8–3.6 mm. Phases, but
not amplitudes, were corrected by an appropriate calculation of the contrast
transfer function. Uncomplexed West Nile virus was used as a starting model
for image reconstruction. The orientations of the particles were determined
using the program SPIDER45. The three-dimensional electron density map was
calculated using the program XMIPP46, which had been modified to handle
icosahedral symmetry. Out of a total of 2,885 boxed particles, 2,186 were used
for the reconstruction of the 37 1C data to achieve a resolution of 24 Å,
determined by dividing the particles into two equal sets and noting the
resolution at which the Fourier shell correlation coefficient fell below 0.5
(Fig. 3a and Supplementary Table 2a, data set 3). The two leaflets of the lipid
bilayers in the viral membrane were not resolved from each other, suggesting
some heterogeneity in the sample.
To verify the validity of the reconstruction, a cryoEM density map generated
from 532 particles using the sample incubated at room temperature
(Supplementary Table 2a, data set 1) was compared with a reconstruction
using a 540-particle subset of the 37 1C data (Supplementary Table 2a,
data set 2). These two maps showed similar density distributions corresponding to a Fourier shell correlation greater than 0.5 at a resolution
of 38 Å, similar to the resolution for both B500 particle data sets (Supplementary Table 2a).
Accession codes. Coordinates for crystal forms 1 and 2 of the Fab 1A1D2:
E-DIII complexes have the PDB accession codes 2R69 and 2R29, respectively.
The cryoEM map of the Fab complexed with native DENV has been deposited
with The European Bioinformatics Institute and has the accession code
EMD-1418. The coordinate for the fitted Fab and E molecules into the cryoEM
map has the PDB accession code 2R6P.
Note: Supplementary information is available on the Nature Structural & Molecular
Biology website.
ACKNOWLEDGMENTS
We thank K. Choi for help with the crystallography, W. Zhang for advice
concerning the cryoEM reconstruction and P.G. Leiman for help in data
collection. We also thank Argonne National Laboratory and the Advanced Photon
Source (Argonne, Illinois, USA) staff at beamline GM/CA 23 for help and
encouragement with diffraction data collection. Use of the Advanced Photon
Source was supported by the US Department of Energy, Office of Science, Office
of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. The GM/CA
CAT is funded by the US National Institute of General Medical Science (Y1-GM1104) and the US National Cancer Institute (Y1-CO-1020). We thank S. Johnson
for help with the sequencing of the 1A1D-2 VH and VL domains. The work was
supported by the Pediatric Dengue Vaccine Initiative (TR-17) to R.J.K. and
M.G.R. and a US National Institutes of Health (NIH) National Institute of
Allergy and Infectious Diseases program project grant (AI055672) awarded to
R.J.K., M.G.R. and others. M.S.D. and D.H.F. were supported by the Midwest
Regional Center of Excellence for Biodefense and Emerging Infectious Disease
Research (V54 A1057160). R.J.K. and M.G.R. also had support from Region V
Great Lakes Regional Center of Excellence (NIH award U54 AI057153).
5
ARTICLES
AUTHOR CONTRIBUTIONS
S.-M.L. prepared Fab fragments and viruses, conducted the neutralization and
temperature experiments and solved both the crystal and cryoEM structures of
the Fab complex. V.K. developed software for the cryoEM reconstruction. G.E.N.
and D.H.F. provided E-DIII. H.A.H., A.J.B. and P.R.C. took the cryoEM images.
J.T.R. and D.S. provided the hybridomas and produced antibodies, respectively.
S.S.-P. and M.S.D. mapped Fab 1A1D-2 epitope on E-DIII by yeast expression
system. R.J.K. initiated the project, established collaborations and advised on
writing of the paper. M.G.R. supervised the project and, together with S.-M.L.,
wrote the paper.
Published online at http://www.nature.com/nsmb/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
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doi:10.1016/j.jmb.2007.08.067
Available online at www.sciencedirect.com
The Three-dimensional Structure of Genomic RNA in
Bacteriophage MS2: Implications for Assembly
Katerina Toropova 1 , Gabriella Basnak 1 , Reidun Twarock 2
Peter G. Stockley 1 ⁎ and Neil A. Ranson 1 ⁎
1
Astbury Centre for Structural
Molecular Biology, University of
Leeds, Leeds, LS2 9JT, UK
2
Departments of Biology and
Mathematics, University of
York, York YO10 5DD, UK
Received 29 June 2007;
accepted 23 August 2007
Available online
7 September 2007
Using cryo-electron microscopy, single particle image processing and threedimensional reconstruction with icosahedral averaging, we have determined the three-dimensional solution structure of bacteriophage MS2
capsids reassembled from recombinant protein in the presence of short
oligonucleotides. We have also significantly extended the resolution of the
previously reported structure of the wild-type MS2 virion. The structures of
recombinant MS2 capsids reveal clear density for bound RNA beneath the
coat protein binding sites on the inner surface of the T = 3 MS2 capsid, and
show that a short extension of the minimal assembly initiation sequence that
promotes an increase in the efficiency of assembly, interacts with the protein
capsid forming a network of bound RNA. The structure of the wild-type
MS2 virion at ∼9 Å resolution reveals icosahedrally ordered density
encompassing ∼90% of the single-stranded RNA genome. The genome in
the wild-type virion is arranged as two concentric shells of density,
connected along the 5-fold symmetry axes of the particle. This novel RNA
fold provides new constraints for models of viral assembly.
© 2007 Elsevier Ltd. All rights reserved.
Edited by J. Doudna
Keywords: cryo-electron microscopy; MS2; ssRNA virus; genomic RNA
structure; atomic structure fitting
Introduction
All viruses face a common challenge of packaging
their nucleic acids into a protective shell of protein
subunits. Different viruses have evolved very
different strategies to permit packaging of either
DNA or RNA genomes whose functions at various
stages of the viral lifecycle require them to adopt
radically different conformations. The stratagem
that many single-stranded (ss)RNA viruses have
evolved is to assemble their capsids around their
genomes. However, a major unresolved problem in
structural virology is to understand the detailed
molecular mechanism(s) that gives rise to the
assembly of capsids of the correct size and symmetry. This is especially true for viruses whose capsids
exhibit quasi-equivalent symmetry in their coat
protein lattices.1 Various proposals for formation
*Corresponding authors. E-mail addresses:
p.g.stockley@leeds.ac.uk; n.a.ranson@leeds.ac.uk.
Abbreviations used: CP, coat protein; TR,
RNA stem–loop; cryo-EM, cryo-electron microscopy.
of protein shells based on the initial formation of
3-fold,2 or 5-fold3 assembly initiation complexes
have been made previously. Capsid assembly is,
however, spontaneous and usually very rapid, with
the result that it has been technically difficult to
isolate and characterise intermediates on the pathway to the final products beyond these “initiation
complexes”.4,5
The RNA bacteriophage MS2 is an ideal model for
investigating such phenomena owing to the extensive biochemical and structural information that is
available. 6–11 We have therefore undertaken a
structural study of MS2 in order to address the
specific question of the role(s) if any of the ssRNA
genome during assembly. MS2 is a member of the
Leviviridae family of viruses that infect male Escherichia coli cells via an initial attachment to the
bacterial F-pilus. It has a single-stranded, positivesense RNA genome of 3569 nucleotides that encodes
just four gene products: coat protein (CP), replicase,
lysis and maturation protein. CP is the most highly
expressed of these four gene products and 180
copies assemble to form a T = 3 icosahedral protein
shell that encapsidates the genome in the mature
0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
The Structure of Genomic RNA in Bacteriophage MS2
virion. A single copy of the maturation protein is
also incorporated into the virion and functions by
binding the F-pilus. The maturation protein–genomic RNA complex is the only viral component to
enter host cells during infection.12
The crystal structure of the wild-type MS2
bacteriophage has been determined to 2.8 Å
resolution.9,13 The CP fold was unique amongst
known icosahedral viruses at that time, although it
has since been shown to be shared by the other
Leviviridae coat proteins.10,14 The main-chain folds
into a five-stranded antiparallel β-sheet with two
antiparallel β-strands folding over it at the N
terminus, and with a kinked α-helix at the C
terminus (Figure 1). The C-terminal α-helices of
two CP monomers interdigitate to form noncovalent dimers (CP2) in the capsid and these can
be isolated as dimers in solution by acid dissociation.7,15 In the T = 3 capsid, the CP is found in three
distinct conformations, termed A, B and C, consistent with the quasi-equivalent symmetry required to
construct a T = 3 structure. The main site of variation
between conformers is in the loop between the F and
G β-strands, which is extended in A and C conformers, but bent back towards the main body of the
subunit in the B conformer (Figure 1(a) and (b)). The
capsid thus contains two types of dimer; A/B and
C/C and the positioning of these different building
blocks within the protein shell controls the size and
symmetry of the viral particle.
Capsid re-assembly can be triggered in vitro by a
sequence-specific RNA–protein interaction between
825
coat protein dimers and an RNA stem–loop (TR) of
just 19 nt derived from the genomic sequence that
encompasses the start codon of the viral replicase.16,17 RNA–protein binding thus achieves two
functions via a single molecular recognition event:
translational repression of replicase and creation of
an assembly competent complex on viral RNA. We
have shown recently by NMR that TR-binding to
CP2 causes an allosteric conformational change
within the protein from a largely symmetrical
structure to an asymmetric one. Using mass spectrometry and size exclusion chromatography, it was
also possible to show that the TR:CP2 complex is
kinetically trapped and forms capsid shells only
very slowly. In contrast, when this complex is added
to excess RNA-free protein, T = 3 shells form rapidly,
implying that the differing dimer conformers
identified by NMR represent A/B and C/C-like
species. Under these conditions the unit of capsid
growth is a coat protein dimer and the intermediates
in the assembly pathway are dominated by the
formation of a species encompassing the first 3-fold
axis of the particle.18 Formation of the 3-fold axis
with its interdigitated A/B and C/C coat protein
dimers commits the phage to assemble into a T = 3
shell.
This is one of the most detailed molecular models
for the switching of quasi-equivalent conformations
available for any viral system. Allosteric, RNAinduced conformational switching, however, raises
the question of how this effect is propagated during
packaging of genomic RNA in which there is only a
Figure 1. Quasi-equivalence in the capsid of bacteriophage MS2. The structures of the MS2 coat protein dimer (CP2) in
the (a) A/B and (b) C/C conformations are shown. The dimers are viewed from the outside, looking towards the centre of
the particle, and the different conformers are coloured blue (A), green (B) and red (C). The main difference between
conformers is in the orientation of the FG-loops (labelled). (c) The T = 3 icosahedral assembly of 60 copies of the A/B dimer
and 30 copies of the C/C dimer. The bent conformation of the FG-loop in the B conformer permits the A/B dimer to fit
around the icosahedral 5-fold axes, whilst extended FG-loops of the A and C conformers interdigitate around the
icosahedral 3-fold axes. All panels for Figures 1 and 2 were produced using PyMOL [www.pymol.org].
826
single occurrence of the TR sequence. We have
shown that the kinetics of assembly are influenced
by short extensions of the TR sequence with the
natural genomic sequence, and that these effects are
sensitive to the polarity of the added sequences.19 It
is therefore important to understand the structure of
the genomic RNA within the viral particle and the
influence of sequences outside of the TR stem–loop
on the assembly pathway.
Here we used cryo-electron microscopy (cryoEM), single particle image processing and 3-D
reconstruction with icosahedral averaging to determine the solution structures of T = 3 shells reassembled with short genomic RNA fragments, and
extend significantly the resolution of a previously
reported solution structure for the wild-type
bacteriophage MS2.20 The resulting structure for
the MS2 virion, at a resolution of ∼9 Å, provides
novel insights into the organization of genomic
RNA. The structure reveals a T = 3 icosahedral
protein shell within which icosahedrally ordered
density for the majority of the genomic RNA is
observed in an unprecedented structure consisting
of two discrete but connected shells of density. The
structures of the complexes with genomic RNA
fragments are consistent with the idea that genomic
RNA follows a defined path with respect to the coat
protein shell and can participate actively in the
assembly pathway.
Results
The structure of recombinant MS2 capsids
reassembled with short genomic RNA fragments
We, and others,16,17 have shown that in vitro
reassembly of T = 3 MS2 capsids can be triggered by
incubation of coat protein dimers with the TR 19
nucleotide stem–loop.18,19 We have also demonstrated that a 5′ extension of TR with 12 nt of genomic sequence (oligonucleotide S2) results in altered
kinetics and yield of capsid formation in in vitro
reassembly assays. This result suggests that
sequences outside the minimal assembly initiation
stem–loop play a role(s) in the assembly pathway.19
To test this idea, we carried out reassembly reactions
with both TR and S2 RNA oligonucleotides at an
RNA:coat protein dimer molar ratio of 1:2 under
conditions known to favour efficient production of
the T = 3 shell in vitro.18 The reassembly reactions
were allowed to proceed for 3 h and then the
resultant products were vitrified and examined in
the electron microscope. Intact, reassembled capsids
are the predominant species in solution after this
period and comprise a relatively homogeneous
population of particles that are readily identified
and selected for image processing and structure
refinement.
The starting model for our reconstructions was the
crystal structure of the MS2 capsid lacking any
coordinates for RNA,13 with all high-resolution
features removed by application of a low-pass
Fermi filter in Fourier space. The inside of such a
starting model is therefore smooth and relatively
featureless, with no protrusions of density into the
encapsidated space (Figure 2(a) and (b)). The refined
structure of the MS2 capsid assembled in the
presence of TR RNA (TR–MS2) shows a strikingly
different picture. Figure 2(c)–(e)) shows the solution
structure of TR–MS2 at ∼16 Å resolution. It can
readily be seen that significant extra density is
present in the map compared to Figure 2(a) and (b).
This density protrudes from the inside surface of the
capsid towards the centre of the particle. In order to
understand the positions of these densities in
relation to the overlying lattice of CP2, we docked
the atomic coordinates for the MS2 capsid into our
cryo-EM density. This (Figure 2(e)) clearly confirms
that the extra densities are located beneath the CP2
sites where the TR operator is known to bind, i.e.
across the ten-stranded antiparallel β-sheet. We
therefore attribute these densities to bound TR
RNAs. Owing to the icosahedral averaging used to
calculate the reconstruction, all CP2 binding sites are
occupied in the density map. The relative strength/
significance of the protein and RNA electron
densities, together with the biochemical observation
that CP2 binds TR with high affinity suggest that the
majority of such sites are occupied in solution. This
is surprising given the stoichiometry of the assembly
reaction, but it suggests that assembling intermediates bind stem–loops with higher avidity than
coat protein dimers. This is consistent with the
observation that TR stem–loops soaked into crystals
of T = 3 recombinant shells that do not contain
tightly bound RNA fragments, bind to both A/B
and C/C dimers.10,21
The averaged electron densities below the A/B
and C/C dimers are different and two modes of
binding are expected based on the crystallographic
data. At the symmetric C/C dimer TR RNA binds
in two symmetry-related orientations.10 Within an
A/B dimer, however, the conformation of the FGloop in the B subunit appears to restrict the binding
of the TR RNA so that at these sites it is found in a
single orientation.10,13 In our icosahedrally averaged
reconstruction, an averaging of the two conformations occurs resulting in a larger density beneath the
C/C dimer compared to that found bound to A/B
dimers.
The close similarity of our EM density to published crystallographic structures indicates that
cryo-EM reconstructions of reassembled MS2 capsids accurately reflect their structure. We therefore
have confidence that the reconstruction with the
longer S2 RNA reveals the path taken by the additional genomic RNA sequence in the wild-type
phage particle. The solution structure of S2–MS2
at ∼18 Å resolution is shown in Figure 2(f)–(h). As
expected, the TR and S2–MS2 reconstructions are
essentially identical in the protein capsid region. The
densities corresponding to bound RNA in the
S2–MS2 map are larger, and encompass the densities
observed in the TR structure, confirming the
827
Figure 2. Solution structures of MS2 capsids containing TR and S2 RNAs. The rear half (a) and a 30 Å thick central
section through the density (b), of the RNA-free MS2 capsid crystal structure, converted to density at ∼20 Å resolution.
The RNA-free atomic coordinates have a smooth inner surface with no significant protrusions of density into the centre of
the particle. (c) The sequences of the TR and S2 RNA oligonucleotides. The same views as shown for the RNA-free MS2
capsid ((a) and (b)) are shown for reassembled TR–MS2 ((d) and (e); pink) and S2-MS2 ((g) and (h); blue-grey).
Reassembled RNA-containing capsids have distinct protrusions of density from the inner surfaces of the capsids,
corresponding to bound RNA. (f) and (i) Difference density maps for TR–MS2 and S2–MS2 were generated by subtraction
of the RNA-free crystallographic density from the respective cryo-EM maps. The density for RNA lying below the A/B
and C/C dimers is shown (inset) for both TR–MS2 and S2–MS2. The crystallographic coordinates are shown in cartoon
representation and coloured as for Figure 1. The resolution of the TR–MS2 and S2–MS2 maps was 16 Å and 18 Å,
respectively, at a Fourier shell correlation (FSC) of 0.5 (see Supplementary Data).
interpretation that they represent RNA. The S2–MS2
map shows that the 5′ extensions of RNA interact
with the protein lattice, forming the beginnings of a
network of bound RNA, rather than extending into
the centre of the particle and being smeared out by
disorder and symmetry averaging. This observation
is consistent with the network of genomic RNA
found beneath the protein capsid for wild-type MS2
phage and other members of the Leviviridae.20,22 It is
also consistent with the idea that these genomic
sequences can play an active role(s) during the
assembly process.
In both reconstructions the centres of the particles
appear free of density. In this in vitro system where
the only nucleic acids present are the oligonucleotides added, no artefactual density from symmetry
averaging of noise was observed except at extremely
low σ values, where disconnected noise appeared.
This noise could be selectively removed using a lowpass Fourier filter. Indeed this was also the case in a
further control where reconstructions were calcu-
lated from a test dataset derived from re-projections
of a low-pass filtered, RNA-free crystal structure to
which Gaussian noise was computationally added
(not shown). The result with S2 oligonucleotides
therefore suggests that the 5′ extended sequences as
well as the minimal TR sequence interact with coat
protein in the capsid, as expected from the earlier
biochemical experiments.
The structure of wild-type bacteriophage MS2
Given the above result for recombinant MS2
capsids assembled with S2 RNA, it seemed likely
that higher resolution information for the wild-type
MS2 virion would be even more informative about
the path of the genomic RNA across the inner
surface of the protein shell. We therefore performed
a new reconstruction of wild-type MS2 virions
which yielded an electron density map at ∼9 Å
resolution. This structure, which represents a dramatic improvement in the available resolution for
828
the intact virus in solution, was calculated from 9335
images, which together with the icosahedral symmetry used for 3-D reconstruction equates to the
averaging of ∼560,000 asymmetric units.
Consistent with previous X-ray crystallographic
and lower-resolution cryo-EM studies, the reconstruction contains clearly resolved density for the
T = 3 icosahedral coat protein lattice, with large
pores through the shell at both the 3 and 5-fold
icosahedral symmetry axes (Figure 3). To aid in the
interpretation of this structure, the atomic coordinates for the MS2 capsid complexed with the TR
oligonucleotide were docked into the EM density
using the fitting software SITUS.23 The atomic
coordinates fit the cryo-EM density extremely
well, although the tips of the loops joining the
first two β-strands (the AB-loop) in the A (shown in
blue) and C (shown in red) conformers protrude
somewhat from the cryo-EM map around the 3-fold
axes. In previous cryo-EM reconstructions (including the TR– and S2–MS2 structures described
above) density for this loop was not seen, yielding
relatively smooth reconstructions which contrast
starkly with available crystallographic structures
where these loops project from the surface of the
capsid giving MS2 a characteristic “spikey” appearance. The crystallographic B-factors for this loop are
significantly higher than for the body of the protein
(with the exception of the FG-loop), and are somewhat higher for this region in conformers A and C
than in conformer B.13 This suggests both that the
AB-loop is flexible and that it is somewhat more
ordered in the B conformer. In our reconstruction
we see these differences reflected in the cryo-EM
map, with the loops largely out of the density for
coat proteins in the A and C conformations,
whereas in the B conformers only the very tips of
the AB-loops protrude from a pronounced surface
feature (Figure 3(e)). This implies that the loops are
more ordered in the B quasi-equivalent subunits
than in A and C, a context dependent consequence
of quasi-equivalence.
The organisation of genomic RNA in WT-MS2
Whilst the cryo-EM structure of the MS2 protein
capsid itself is consistent with previous studies,
when the interior of the virion is examined entirely
new features are observed. Figure 4 shows difference density maps where the cryo-EM density
corresponding to the capsid region has been
selectively removed, leaving only the density
features within the interior of the capsid. Sections
through this masked WT–MS2 reconstruction,
together with the atomic coordinates for the capsid,
are shown perpendicular to the icosahedral 2-fold,
3-fold and 5-fold axes (Figure 4(a)–(c), respectively).
Substantial ordered density, representing the encapsidated, genomic RNA occupies the interior of the
virion as two connected shells (see Supplementary
Data, movie 1). The outermost shell lies directly
beneath the protein capsid between (averaged) radii
of ∼108 Å and ∼84 Å. In agreement with previous
EM20 and crystallographic studies with short RNA
Figure 3. The solution structure of the wild-type bacteriophage MS2 virion. (a) Surface view and atomic structure
fitting of the WT–MS2 cryo-EM structure. On the left, the cryo-EM density envelope is shown as a solid grey surface. On
the right, the fitted atomic coordinates ((PDB ID: 1aq324) are included in a cartoon representation (coloured as for
Figure 1), embedded within a transparent surface. The resolution of the map was 8.9 Å (FSC = 0.5; see Supplementary
Data). (c)–(e) Details of the fit of atomic coordinates into the WT–MS2 structure. The views shown are perpendicular to the
(b) 2-fold, (c) 3-fold and (d) 5-fold global symmetry axes. (e) The different quasi-equivalent environments for the A, B and
C coat protein conformers. The upper portions of the AB-loops in conformers A and C do not fit into the EM density,
whilst for conformer B, all but the very tip of this loop is buried in a pronounced surface feature (in the foreground of the
Figure). All images in Figures 3–5 were created using UCSF Chimera [http://www.cgl.ucsf.edu/chimera].
829
Figure 4. The organisation of genomic RNA in the WT–MS2 virion. The 40 Å thick central sections through the virion
are shown, perpendicular to the (a) 2-fold, (b) 3-fold and (c) 5-fold icosahedral symmetry axes. The crystallographic
coordinates for the WT–MS2 protein shell (in cartoon representation and coloured as for Figures 1–3) are shown in each
view with regions of the cryo-EM map at lower radii shown as radially coloured density (from pale blue (r ∼108 Å) to pale
red (r ∼42 Å)). The cut surface of the EM density is shown in the same colour scheme.
fragments,10,24 this shell consists of a connected
network of density that maps the binding sites for
RNA on the inner surface of the coat protein lattice.
The second density shell is found at much lower
radii, occupying a region extending from ∼42-65 Å
from the centre of the virion.
Around the icosahedral 5-fold axis, weak connections are made from the protein-bound RNA layer,
to a substantial density that extends down the 5-fold
axis for more than 40 Å towards the centre of the
virion. These densities run from the edge of the
outer RNA shell (r ≈ 82 Å) to the innermost edge of
the inner shell (r ≈ 40 Å). The volume of the major
densities lying along the 5-fold axes appears only
large enough to encompass a single duplex of RNA.
At low radii (40–50 Å), further connections between
the densities on the 5-fold axes are seen. At radii
below the innermost edge of the inner shell, the
centre of the virion is empty.
There are no features in the icosahedrally averaged cryo-EM density map that can be assigned to
the single copy of the maturation protein present in
the virion. Since this protein must be located at least
in part at the surface of the phage particle, it is
possible that some of the density along the 5-fold
axis arises from inappropriate icosahedral averaging
of this protein.
The combined volume of the density that we
ascribe to RNA accounts for ∼25–30% of the internal
volume of the capsid. Owing to the wide range of
published estimates for the density of RNA, the
fraction of the genomic material encompassed by
this density is difficult to estimate accurately. However, using the volume occupied by the nucleotides
of TR seen in crystallographic studies as a guide, we
estimate that the encapsidated density we attribute
to RNA may accommodate ∼90% of the 3569
nucleotides in the wild-type MS2 genome. Given
the significant and obvious errors implicit in such a
calculation, there remains the possibility that the
entire MS2 genome may be packaged with nearicosahedral order at this resolution, an unprece-
dented degree of ordering for a single-stranded
RNA virus.
Binding of genomic RNA to the inner surface of
the WT-MS2 capsid
Figure 5 shows details of the interaction between
the genomic RNA density and the inner surface of
the WT–MS2 capsid. The density in this RNA shell
is the result of icosahedral symmetry averaging.
Nevertheless the RNA density below each type of
quasi-equivalent coat protein dimer is different. For
dimers in the A/B conformation, density for bound
RNA is observed beneath the dimer interface and
extends towards the icosahedral 5-fold axes. These
axes are surrounded by dimers in the A/B conformation, and so a connected ring of density is
formed encircling each 5-fold axis. Weak density
connections are made from this ring to the inner
shell of RNA. The RNA density beneath the C/C
dimers also sits beneath the dimer interface, but the
connectivity is different to that at the A/B site
(Figure 5), suggesting that the average interaction
between coat protein and genomic RNA is different
at these positions. This is precisely what the allosterically mediated model of the assembly pathway
predicts.
Discussion
The range of different solutions that viruses have
evolved to overcome the challenge of packaging
their genomic material is very broad. Doublestranded (ds)DNA viruses use powerful packaging
motors that actively insert their base-paired genomes into pre-formed pro-capsids. Such active
packing typically results in a close-packed, spooled
array of DNA duplexes with little if any free volume
remaining within the viral capsid. At the other
extreme, many RNA viruses assemble their capsids
spontaneously, coincident with folding of their
830
Figure 5. Non-sequence-specific binding of genomic RNA to A/B and C/C dimers in the WT–MS2 capsid. (a) An
oblique view of the inner surface of the WT–MS2 capsid. The relative positions of the icosahedral 2-, 3- and 5-fold axes are
indicated by a dotted black line, a black triangle and a black pentagon, respectively. The density for the cryo-EM map is
rendered as a grey surface, with the density lying directly beneath an A/B dimer shown in blue, and that below a C/C
dimer highlighted in purple. (b) Close-up view of the RNA density (in blue) beneath an A/B dimer (shown in cartoon
representation and coloured as for all other Figures). (c) Crystallographic structure of the TR oligonucleotide bound to an
A/B dimer. The bound RNA is shown as an orange ribbon (1AQ319). (d) Close-up view of the RNA density (in purple)
beneath a C/C dimer. (e) Crystallographic structure of the two symmetry-related binding modes for TR beneath the C/C
dimer.
single-stranded genomes.2,25,26 However, the mechanisms by which capsid assembly and genome folding
operate and interact remain poorly understood.
The structure of the MS2 genome in the viral
capsid
The reconstruction of the wild-type MS2 phage
particle at intermediate resolution (∼9 Å) presented
here gives a unique insight into the structure and
organization of the viral genome. In contrast to the
dsDNA viruses discussed above, the MS2 virion is
remarkably empty. Only approximately one-quarter
of the encapsidated volume of the bacteriophage is
occupied with density for RNA, which together with
the substantial pores through the capsid structure at
both the 3- and 5-fold symmetry axes, suggests that
those portions of the folded RNA molecule not
bound to protein will be fully hydrated.
X-ray crystallography of the same bacteriophage
at 2.8 Å resolution showed no density for the
RNA component other than weak, disconnected
density ascribed to a purine below the known TR
adenine (A-4) binding pocket residues.9 This difference suggests that the icosahedral ordering seen in
the cryo-EM density does not extend to high resolution. The relative electron density of the RNA in
the two inter-connected shells is similar in some
areas to the levels observed for the coat protein
shell, suggesting that these features in the RNA
reflect relatively full occupancy and do not simply
represent an average of several differing conformations within the capsid. In contrast there is a
clear weakening of the density of the connectors as
they extend towards the outer RNA shell. This is
consistent with averaging around the overlying
5-fold axis in the particle, whilst the major connections to the inner shell remain strong. The
implication is that RNA is transiting between shells
from different points around the outer 5-fold axis
but ending up in a unique position on the inner
shell. Such an inter-connected pair of shells of
genomic RNA density has not been observed previously, although this type of organization may be
fairly common. For instance there is a well-ordered
dodecahedral cage of RNA located underneath the
protein shell of Pariacoto virus, but this only
accounts for some 35% of the genomic RNA, the
remainder presumably being located at lower radii
but not visible in either X-ray or cryo-EM maps.27,28
The high degree of ordering of the MS2 genome at
this resolution is of interest because unlike many
other ssRNA viruses the MS2 coat protein lacks
N-terminal extensions (or “arms”) that can interdigitate with the packaged genome and thus
propagate the icosahedral symmetry of the protein
shell throughout the particle. In the case of MS2 the
ordered structure observed must arise principally as
a consequence of RNA folding. There are, however,
several sequence-specific contacts to protein components of the virion that must also help to position
defined RNA sequences within this fold(s). These
are the assembly initiation complex made by the
coat protein with the TR stem–loop and the contacts
to sequences located towards the genomic 5′ and 3′
ends that are made by the single copy of maturation
protein.29 Given the locations of these proteins, i.e.
either wholly (CP) or at least partially (maturation
protein) on the outer surface, it is tempting to
speculate that all three of these RNA sequences must
be in the outer shell of RNA. Alternatively if the
maturation protein were spatially extended it could
span all three major molecular layers of the virion
making one or both contacts with the RNA in either
shell. These three specific constraints, as well as the
network of non-sequence specific interactions required to switch quasi-equivalent conformers, and
the need to fold into the double shell structure with
defined size and symmetry, suggest that the observed cryo-EM density corresponds to a relatively
small, well-ordered ensemble of permissible RNA
folds. This proposal is inherently testable and appropriate experiments are underway to examine it.
Implications for MS2 assembly
The structures of the reassembled particles in the
presence of short genomic sequences are consistent
with the inferences made on the basis of biochemical
reassembly assays, namely that genomic sequences
outside the TR stem–loop can influence the assembly pathway via interaction with CP subunits.18,19
We have shown in a TR-induced reassembly
reaction that the TR-bound CP2 is asymmetric and
the complex metastable until excess RNA-free CP2,
which is primarily a symmetric dimer, is added.18
Once both types of dimer are present assembly of
the T = 3 shell is rapid and proceeds by coat protein
dimer addition, apparently through intermediates
that are dominated by stoichiometries corresponding to the formation of the particle 3-fold axis, i.e. 3
(TR + CP2) + 3(CP2). The clear implications are that
the binding of TR RNA converts a CP2 from a C/Clike species to an A/B-like species and that both are
required for efficient assembly. This RNA-induced,
allosteric switching between quasi-equivalent conformers completely explains assembly in the presence of multiple copies of the TR sequence. Similar
assays in the presence of S2 RNA show significant
increases in the rate and yield of assembly.19 These
can now be understood in structural terms, since the
additional nucleotides interact with a neighbouring
coat protein dimer. It is interesting to speculate
whether such interactions are completely indifferent
to the sequence of this RNA extension.
The assembly pathway followed when genomic
RNA is being packaged cannot, however, be
identical to that seen with the TR oligonucleotide.
Genomic RNA sequences play essential roles in
virion assembly, and the RNA molecule must be
compacted to fit within the mature virion. It must
also serve as the message for viral protein synthesis,
and as a substrate for RNA replication. The kinetics
and mechanism of folding this RNA into the twoshell structure are obviously major factors in
compacting the genome to the dimensions of the
eventual T = 3 shell. Whether the RNA folding
occurs before or during capsid assembly is currently
unknown, although the latter mechanism would be
akin to the assembly of the ribosome, another
complex containing both protein and large, complex
and highly folded RNA molecules. What is clear
from the cryo-EM reconstruction is that networks of
831
RNA–protein contacts are created throughout the
virion that are, on average, distinct at A/B and C/C
dimers. This observation is consistent with the idea
that sequences/secondary structures in the genome
other than TR can contribute to quasi-equivalent
conformer switching at defined positions throughout the assembly process. Presumably this genomemediated assembly pathway is much more efficient
than reassembly in the presence of short fragments
in sub-stoichiometric amounts which is also known
to occur.
Such an RNA-templated capsid assembly pathway has a number of features that constrain the
RNA folding even further. Larson & McPherson30
have addressed this question for satellite tobacco
mosaic virus (STMV), a T = 1 structure where up to
80% of the genomic ssRNA can be accounted for as
icosahedrally ordered stem–loops located in a single
shell complexed with the coat protein. They argued
that the STMV RNA packaged in the virion is likely
to form secondary structures dominated by local
stem–loop interactions formed as the genomic
transcript emerges from a replication complex.
Such local structural motifs are separated by
single-stranded regions that allow the RNA a great
deal of conformational freedom, permitting local
sequence/structure variations to occur whilst still
fulfilling the need to make interactions with each
facet of the protein lattice. In this model the
packaged RNA structure need not correspond to
the lowest free energy or physiologically active
forms of the molecule.
The situation must be different in MS2 for two
reasons. Firstly we have successfully reassembled
T = 3 shells from in vitro transcribed and purified
large sub-genomic fragments of RNA that have had
the opportunity to form long range secondary and
tertiary interactions (unpublished data). Secondly
the formation of a double-shell structure of RNA
imposes additional constraints on the path of the
RNA within the virion. A useful way to think about
this problem is to project the outer RNA network
onto a plane net of the protein shell (Figure 6).1
Figure 6(a) shows the locations of the capsid
protein dimers relative to the surface of an icosahedron in a planar representation. This is a schematic
view of the spherical arrangement in Figure 6(b) and
facilitates comparison with the location of the RNA
density (Figure 6(c)). The RNA within the outer shell
forms a polyhedral cage (Figure 6(d)) with 60
vertices located underneath the 60 A/B dimers.
The contacts between RNA and A/B dimers are
important for conformational switching from a
symmetric to the required asymmetric form, implying that the RNA meets every vertex of this
polyhedral cage. In contrast, this model does not
require the path of the RNA to traverse all the edges
of the polyhedron, which are positioned underneath
the C/C dimers. The RNA density observed beneath
C/C dimers could result from secondary structure
elements arising at either vertex bordering that edge.
We also know that capsid assembly takes place by
addition of coat protein dimers to the growing
832
Figure 6. The relationship of capsid protein dimers to the RNA density in the outer shell. (a) The A/B and C/C dimers
relative to the surface of an icosahedron that has been flattened onto a plane. Coat protein subunits in the A, B and C
conformers are indicated by lettering, colour-coded as for previous Figures. Coat protein dimers surrounding one
icosahedral 3-fold axis have been highlighted with a coloured background. (b) The corresponding spherical
representation to the planar representation shown in (a). (c) The location of RNA density superimposed on the coat
protein lattice, where the path taken by the genomic RNA molecule is indicated by a magenta line. (d) A threedimensional, polyhedral representation of the plan schematic shown in (c).
intermediates, with A/B dimers resulting from
binding to (stem–loops of) RNA. Due to the
requirement to make these RNA–protein interactions, the formation of the folded RNA shell and of
the protein lattice are likely to occur simultaneously.
Folding of the RNA into secondary and tertiary
structures appropriate for packaging, if necessary,
may be facilitated in part by the interaction with coat
protein. As Figure 6(c) shows, even if assembly is
initiated at a single point (TR) on the RNA there are
multiple sub-pathways that intermediates could
then follow to reach the final T = 3 capsid.
There could be further constraints implied by the
connections between the inner and outer RNA
cages. As shown in Figure 4, the two shells of
RNA density are connected along the particle's
5-fold axes. The observed density of these connections is consistent with the presence of at most a
single RNA duplex or perhaps two single strands of
RNA (Figure 7(a)) at every 5-fold axis. If we consider the path of the genomic RNA around the outer
shell, at some point it must cross to the inner shell
and then connect back to the outer shell. The two
possible ways in which these transitions could occur
are shown in Figure 7(b) and (c). In the first of these,
the RNA strand leaves the outer shell, and returns
to it, at the same 5-fold axis (Figure 7(b)). The
alternative is that RNA strand leaves at one 5-fold
axis and returns at a different 5-fold axis, i.e. that it
leaves at one vertex of the outer shell and returns to
a neighbouring position (Figure 7(c)). If the latter
were correct, a single strand RNA cannot account
for the observed density, so a second strand would
be required to transit in the opposite direction at the
original 5-fold axis after following a distinct pathway. Moreover, the single RNA strand would first
have to transit back to the outer shell at a different
5-fold axis, with no obvious constraints on the location of that axis. This seems highly unlikely given
the efficiency of capsid assembly and the propensity
of RNA to form base-paired duplexes. If the former
were true, the connections and the inner shell could
be composed of discrete secondary structural elements, thus imposing far fewer constraints on RNA
folding and evolution.
Implications for phage infection
RNA phages associate with target E. coli cells by
binding to the F pilus via the maturation protein.12
Only the maturation protein–genomic RNA complex then enters the cell via a mechanism that is
currently not understood. The cryo-EM structure of
the MS2 virion presented here strongly suggests
that, in sharp contrast to double-stranded (ds)DNA
viruses, there is little or no driving force within the
833
Figure 7. Possible modes of connection between RNA shells. (a) A close-up view of the RNA density along an
icosahedral 5-fold axis. The cryo-EM density is shown as a semi-transparent, radially coloured surface. The radial colour
scheme is identical to that shown in Figure 4. An A-type RNA duplex (taken from the crystal structure of Pariacoto virus
(1F8V27) is modelled into the cryo-EM density by hand for scale comparison. Two possible modes of RNA structure are
compatible with this structure. In (b), the RNA strand leaves the other shell near a 5-fold vertex, transits to the inner shell,
and returns to the outer shell at the same 5-fold vertex. In (c), the RNA strand returns to the outer shell at a different 5-fold
axis (in yellow) and must revisit each vertex at a later stage to allow hybridisation of sequences at different points in the
genome into a duplex structure (in red).
capsid to achieve this genome release. The RNA
genome is not close-packed within the virion, and
the capsid is porous to small molecules. This in turn
suggests that the driving force for genome release
must be derived from some function of the host
bacterium. A recent low-angle neutron scattering
study suggested that the dimensions of the MS2
capsid proteins are different in the presence or absence of maturation protein, the form lacking maturation protein being considerably thicker (31–37 Å
versus 21–25 Å) than in wild-type particles.31 This
difference was ascribed to the formation of a postinfection state within the capsid lattice when both
RNA and maturation protein have left the protein
shell. This state was not revealed by previous X-ray
structures, even those of recombinant shells lacking
maturation protein, it was argued because crystallization favours the “thin” pre-infection state. We
see no evidence for two such states in our data. The
solution structures of both reassembled and wildtype phage map onto the X-ray structures of both
phage and recombinant protein shells with high
fidelity.
Conclusions
High-resolution structures for large RNA molecules are rare, and the MS2 genomic RNA structure
presented here is amongst the highest resolution
for such a large RNA molecule (by comparison
the 23S rRNA of the prokaryotic 50S large
ribosomal subunit determined at atomic resolution
is ∼2900 nucleotides in length). Positive sense,
single-stranded RNA viruses are the most common
type of viruses in nature. Unlike many of these,
however, MS2 does not interact with its genome via
extensions (or “arms”) from the body of its coat
protein. The data presented here provide the first
direct evidence that a viral shell lacking such arms
to propagate icosahedral symmetry through the
particle, can promote the folding of a large RNA
molecule into such a compact, ordered fold. Such
large RNA molecules are relatively common and
are of enormous importance in cells. It is known
that many of their functions are regulated by RNA
folding. The double-shell structure seen here may
well represent a general architecture for compacting such large molecules into non-active states.
Materials and Methods
Proteins, RNAs and reagents
Wild-type bacteriophage MS2, maintained in buffer A
(10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 0.1 mM MgCl2
and 0.01 mM EDTA), was a gift from Professor David
S. Peabody, University of New Mexico (Alburquerque).
Wild-type, recombinant MS2 coat protein (CP) was overexpressed in E. coli and purified using described methods.32,33 Purified T = 3 capsids were stored in 10 mM
Hepes (pH 7.2), 100 mM NaCl and 1 mM EDTA. CP2 for
reassembly reactions was obtained by treating the
recombinant capsids with glacial acetic acid to induce
capsid dissociation to disassembled CP monomers,
followed by exchange into 20 mM acetic acid to promote
dimerisation.15 To ensure that complete disassembly had
occurred before reassembly, the CP2 were examined using
negative stain EM. Synthetic oligonucleotides, TR and S2,
were prepared as described.34
Recombinant MS2 reassembly reactions
Capsid reassembly was carried out by mixing TR or S2
with CP2 in a 1:2 molar ratio in buffer B(20 mM Trisacetate, 0.4 mM magnesium acetate, 20 mM acetic acid
(pH 5.2)) on ice. After reassembly for 3 h, samples were
flash-frozen as described below.
834
Cryo-EM data collection and image pre-processing
The 3 μl aliquots of wild-type MS2 at 0.8 mg ml−1 in
buffer A and reassembly reaction samples in buffer B were
placed onto 300-mesh Quantifoil R2/1 grids that were
glow-discharged in air for ∼30 s immediately before use.
Grids were frozen by plunging into liquid nitrogencooled, liquid ethane, using a computer-controlled,
pneumatically driven freezing apparatus.35 Samples
were imaged on an FEI Tecnai-F20 microscope equipped
with a Gatan 626 cryo-transfer stage, using low-dose protocols (∼15 e−/Å2). Wild-type MS2 was imaged on Kodak
SO-163 film at 50,000× magnification and micrographs
were digitized using a Nikon Coolscan3000 scanner at a
final object sampling of 1.25 Å/pixel. Reassembly reactions were imaged on a GATAN US4000SP, 16Mpixel CCD
camera at 50,500× magnification, giving a final object
sampling of 2.97 Å/pixel.
Micrograph defocus and astigmatism were determined
computationally using the program CTFFIND3,36 and
micrographs showing significant drift or astigmatism
were discarded. All particles recognisable as isolated
MS2 capsids were semi-automatically selected using the
program BOXER37 and correction for the microscope
contrast transfer function was performed by computational flipping of image phases in SPIDER.38 All other
image processing steps were performed in SPIDER unless
otherwise stated. For wild-type MS2, 10,382 molecular
views were extracted into 448 × 448 pixel boxes and bandpass filtered between 350 Å and 4 Å. For reassembly
reactions with TR and S2 RNAs, 1550 and 1715 molecular
views, respectively, were extracted into 320 × 320 pixel
boxes and band-pass filtered between 350 Å and 6 Å. All
extracted images were normalised to a constant mean and
standard deviation.
Single particle reconstruction
The atomic coordinates for wild-type MS2 protein
capsid (PDB ID: 2ms213) were converted to density and
all high resolution features removed by Fourier filtration
to 40 Å using a low-pass Fermi filter. This low-resolution,
RNA-free representation of the MS2 capsid was then used
as a starting model for EM structure refinement of all the
data. For wild-type MS2, the low-resolution density was
projected across 78 orientations to give a range of molecular views evenly covering the icosahedral asymmetric
unit with a spacing of 3°. All image data were then
iteratively aligned to these views. Following each alignment, averages of the images corresponding to each view
were calculated. 3-D reconstructions were generated after
each refinement round using weighted back-projection
and imposing icosahedral symmetry. The resulting reconstruction was then used to generate a new series of
reference views, after masking with a tight, soft-edged
mask derived from the density map, and the whole
procedure iterated. In the final rounds of refinement,
images were ranked according to cross-correlation coefficient and the worst aligning members for each orientation
were excluded. This was implemented in such a way that
the best-populated molecular view contained no more
than three times as many raw images as the worst
populated view. The resolution of the final reconstruction,
calculated from 9335 raw images, was determined to be
9 Å using the 0.5 Fourier shell correlation criterion (see
Supplementary Data).
For reassembly reactions of recombinant MS2 capsids,
3-D reconstructions were generated using the method
described above, with the exception that the low-resolution starting model was projected across 53 orientations
(angular spacing of 3.5°). Final reconstructions contained
957 and 1152 individual molecular views for TR and S2,
respectively, and the final resolutions were determined to
be 15.5 Å and 17.8 Å, using the 0.5 Fourier shell correlation
criterion (see Supplementary Data).
Masking and RNA volume estimation
To remove density corresponding to the protein shell
from the WT–MS2 cryo-EM map, a mask was made by
taking the crystallographic coordinates for the protein
shell, converting to density and removing high-resolution
features by Fourier filtration to the same resolution as the
cryo-EM map. This density was then converted to binary
and inverted to form a hard-edged mask. The edges of this
mask were then softened with a second Fourier filtration
step using a relatively sharp Fermi filter profile in SPIDER.
Finally, the cryo-EM maps were multiplied by the mask to
remove protein density.
A benchmark figure for the per-base volume of RNA
was estimated by taking the 60 copies of the TR oligonucleotide present beneath A/B dimers in the crystal
structure of the MS2–TR complex (1AQ3), which each
contain 16 ordered nucleotides (960 in total), and converting to density in SPIDER. The resulting density was
Fourier filtered to remove high-resolution information.
The combined volume of these densities, and of the RNA
density in the WT–MS2 structure were then calculated in
UCSF Chimera.
Atomic structure fitting
An initial fit of the MS2 capsid was generated by
manual docking of the atomic coordinates of the MS2
capsid (PDB ID: 1AQ324) into the wild-type MS2 EM
density map using PyMOL†. The orientation of this
manual fit was then refined using the program SITUS.23
This refined orientation was also used for the cryo-EM
map for capsid reassembled with TR, which is in the same
coordinate system owing to the image processing strategy
employed. The cryo-EM maps for WT-MS2, TR-MS2 and
S2-MS2 are deposited in the EMDB as EMD-1431, EMD1432 and EMD-1433, respectively.
Acknowledgements
We thank Professor David Peabody, University of
New Mexico for his gift of wild-type MS2 bacteriophage, Dr Sean Killen and Mike Wallis for
computing support, and Dr Abdul Rashid for
oligonucleotide synthesis. We thank Ottar Rolfsson
for discussion of unpublished data. K.T. is a research
student supported by the University of Leeds
Interdisciplinary Institute (UII) in Bionanosciences,
which also funded the work. R.T. is an EPSRC
Advanced Research Fellow and P.G.S. acknowledges financial support from the UK BBSRC and
† DeLano, W. L. (2006). PyMol, DeLano Scientific;
http://pymol.sourceforge.net/
The Leverhulme Trust. N.A.R. holds a University of
Leeds Research Fellowship.
Supplementary Data
Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.jmb.2007.08.067
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SEE COMMENTARY
Near-atomic resolution using electron cryomicroscopy
and single-particle reconstruction
Xing Zhang*, Ethan Settembre†, Chen Xu‡, Philip R. Dormitzer†§, Richard Bellamy¶, Stephen C. Harrison†!**,
and Nikolaus Grigorieff*,**
*Department of Biochemistry and Howard Hughes Medical Institute, and ‡Rosenstiel Basic Medical Sciences Research Center, Brandeis University, MS029,
415 South Street, Waltham, MA 02454; †Laboratory of Molecular Medicine, and !Howard Hughes Medical Institute, Children’s Hospital, 320 Longwood
Avenue, Boston, MA 02115; and ¶School of Biological Sciences, University of Auckland, Auckland, New Zealand
Contributed by Stephen C. Harrison, December 10, 2007 (sent for review November 26, 2007)
FREALIGN " virus structure " rotavirus " protein structure "
image processing
C
ryogenic preservation of macromolecular assemblies was
introduced into electron microscopy 30 years ago (1, 2).
Different applications of electron cryomicroscopy (cryo-EM)
include 2D crystallography, helical reconstruction (helices are
effectively rolled-up 2D lattices), single-particle reconstruction,
and tomography (reviewed recently in ref. 3). In several cases,
data collected from 2D crystals and helical specimens have led
to structures at 4-Å resolution or better, enabling their interpretation by atomic models (for example, see refs. 4–6). The
periodicity present in images of 2D crystals and helical specimens generates distinct diffraction patterns with signalcontaining spots and lines that can be separated from the
background. Removal of the background (noise) is straightforward and amounts to averaging over all unit cells in the crystal.
The distinction between signal and background is not possible in
images of single particles and tomographic reconstructions.
Instead, in the case of single-particle reconstructions, averaging
must be performed over many individual particle images. The
resolution obtained by this averaging procedure depends critically on the accuracy with which each particle can be brought
into register with the others. This computational step is not an
issue for well ordered 2D crystals and helical specimens used in
high-resolution studies because intermolecular contacts establish the alignment of molecules. Other important imaging pa-
www.pnas.org#cgi#doi#10.1073#pnas.0711623105
rameters, such as the magnification, defocus, and beam tilt used
to collect the images in the microscope, also are easier to
determine for 2D crystals and helical specimens because a single
image typically contains 100 to 1,000 times more signal than an
image of an individual molecule or complex (3). For example, a
purple membrane crystal containing !140 " 140 unit cells has
a molecular mass of !1,760 MDa (7), generating !700 times the
signal of a bacterial ribosome of 2.5 MDa (8). Finally, in most
cases, a carbon film is used to support the crystalline specimens,
providing additional stability to the specimen that can reduce
movement during exposure to the electron beam (9).
Because of the lack of periodicity and, in many cases, the lack
of a stable support film, single-particle reconstruction techniques have so far fallen short of yielding structures interpretable
by atomic models. Efforts to achieve ‘‘atomic resolution’’ have
continued, however, because the single-particle technique is
applicable to a wide range of noncrystalline samples and can be
used with just a few picomoles of material (10). Furthermore,
single-particle techniques also can be applied to individual unit
cells or mosaic blocks of crystalline samples, where they can
perform better than crystallographic and helical techniques if
the sample is partially disordered (11, 12). The single-particle
technique therefore assumes a central role in cryo-EM. The
cryo-EM-based 3D reconstruction of an icosahedral virus particle described in this article has a resolution adequate for tracing
a polypeptide chain. The result demonstrates directly that recently developed computational techniques (13) can extract
atomic-level detail from images recorded with contemporary
instrumentation.
Rotaviruses are multishelled, nonenveloped, icosahedrally
symmetric viruses with an 11-segment, double-strand RNA
genome (14). Infectious virions are sometimes called ‘‘triplelayer particles’’ because three distinct protein shells surround the
packaged viral RNA. During cell entry, the outer layer is lost,
and the remaining ‘‘double-layer particle’’ (DLP), which contains approximately one copy of the viral RNA-dependent RNA
polymerase and the capping enzyme for each genomic segment,
generates mRNA transcripts and exports them into the cytoplasm of the infected cell. The inner layer of the DLP, which
Author contributions: P.R.D., S.C.H., and N.G. designed research; X.Z., E.S., and C.X. performed research; E.S., P.R.D., and R.B. contributed new reagents/analytic tools; X.Z., C.X.,
S.C.H., and N.G. analyzed data; and S.C.H. and N.G. wrote the paper.
Conflict of interest statement: P.R.D. is currently employed by Novartis Vaccines and
Diagnostics.
Data deposition: Density maps of DLP and the nonicosahedrally averaged VP6 structure
have been deposited in the Macromolecular Structure Database at the European Bioinformatics Institute, www.ebi.ac.uk/msd-srv/emsearch/index.html (accession codes 1460
and 1461, respectively).
See Commentary on page 1779.
§Present
address: Novartis Vaccines and Diagnostics, 350 Massachusetts Avenue, Cambridge, MA 02139.
**To whom correspondence may be addressed. E-mail: harrison@crystal.harvard.edu or
niko@brandeis.edu.
© 2008 by The National Academy of Sciences of the USA
PNAS " February 12, 2008 " vol. 105 " no. 6 " 1867–1872
BIOPHYSICS
Electron cryomicroscopy (cryo-EM) yields images of macromolecular assemblies and their components, from which 3D structures
can be determined, by using an image processing method commonly known as ‘‘single-particle reconstruction.’’ During the past
two decades, this technique has become an important tool for 3D
structure determination, but it generally has not been possible to
determine atomic models. In principle, individual molecular images
contain high-resolution information contaminated by a much
higher level of noise. In practice, it has been unclear whether
current averaging methods are adequate to extract this information from the background. We present here a reconstruction,
obtained by using recently developed image processing methods,
of the rotavirus inner capsid particle (‘‘double-layer particle’’ or
DLP) at a resolution suitable for interpretation by an atomic model.
The result establishes single-particle reconstruction as a highresolution technique. We show by direct comparison that the
cryo-EM reconstruction of viral protein 6 (VP6) of the rotavirus DLP
is similar in clarity to a 3.8-Å resolution map obtained from x-ray
crystallography. At this resolution, most of the amino acid side
chains produce recognizable density. The icosahedral symmetry of
the particle was an important factor in achieving this resolution in
the cryo-EM analysis, but as the size of recordable datasets increases, single-particle reconstruction also is likely to yield structures at comparable resolution from samples of much lower symmetry. This potential has broad implications for structural cell
biology.
Fig. 1. Image of rotavirus DLPs frozen in ice over a hole in C-flat carbon grids.
The arrows indicate particles that differ significantly in appearance from other
particles in the image, indicating partially damaged particles. These particles
were not used for further processing. More subtle damage to particles that is
not readily visible may be one reason for the lower correlation coefficients
with the reference seen for many particles (Fig. 5).
encloses the RNA, contains 120 copies of viral protein 2 (VP2;
102 kDa); the outer layer contains 780 copies of VP6 (41 kDa).
VP6 forms stable trimers, which pack outside the VP2 layer in
a T # 13 icosahedral array. A recently completed x-ray crystal
structure of the DLP has yielded a full atomic model, refined at
3.8-Å resolution (B. McLain, E.S., R.B., and S.C.H., unpublished
data). A previously determined structure of isolated VP6 trimers
provides an even more precise view of that component (15).
Results
Quality of Data. Data were collected from two DLP preparations.
We aligned 18,125 images of rotavirus DLPs frozen in ice (Fig.
1) and merged a subset of these into a 3D reconstruction by using
the computer program FREALIGN (13). Analysis of the aligned
Fig. 3. Density map of part of the VP2 portion of DLP. The map was filtered
at 4.5-Å resolution and sharpened by using a B factor of $450 Å2. The quality
of the map is similar to that of VP6 before nonicosahedral averaging (Fig. 2d).
particles, which have a diameter of !710 Å, revealed a clear
bimodal distribution of correlation coefficients between individual images and the refined reconstruction. A particle parameter search (see Materials and Methods) moved !13% of the
particles from the lower cluster to the cluster with a higher
correlation. This search was computationally expensive and had
to be limited to a repeat of 50 times. The number of moved
particles was found to depend on the number of search orientations, suggesting that one reason for a low correlation coefficient is misalignment. Other reasons may include more subtly
damaged or otherwise disordered particles. An analysis of
averaged power spectra calculated from particles selected from
both clusters showed Thon rings (16) to a resolution of !6 Å,
suggesting that the image quality is comparable in both cases.
Selecting only the 8,400 particles from the cluster with a higher
correlation, we computed a reconstruction with a resolution of
!5 Å (Figs. 2–4). The icosahedral symmetry of the particle was
applied during reconstruction, increasing the effective size of the
dataset 60-fold. We also used the remaining particles to calculate
a second reconstruction (data not shown), which had a resolu-
Fig. 2. VP6 trimers in the DLP. (a) Rotavirus DLP structure filtered at 20 Å. VP6 trimers involved in the 13-fold nonicosahedral averaging are indicated by letters.
The T trimer coincides with one of the icosahedral threefold axes and contains only one unique VP6 monomer. The other four trimers contain the additional
12 VP6 molecules that were used for nonicosahedral averaging. (b) T trimer of VP6, filtered at 7-Å resolution, with a bundle of three generic helices docked into
easily identifiable density features; 12 other bundles were docked in equivalent density in the other four VP6 trimers. (c) Overview of the 13-fold averaged VP6
trimer at 3.8-Å resolution. Different areas are highlighted and labeled with numbers. They are shown in more detail in d and e and in Fig. 6. (d) Density in area
1 before 13-fold averaging, shown with the atomic model of VP6, determined by using the x-ray density map (B. McLain, E.S., R.B., and S.C.H., unpublished data).
(e) Same as in d but after 13-fold averaging. All structures were prepared with UCSF CHIMERA (35).
1868 " www.pnas.org#cgi#doi#10.1073#pnas.0711623105
Zhang et al.
SEE COMMENTARY
Fig. 4. FSC curves before (black) and after (red) 13-fold nonicosahedral
averaging. The black curve suggests a resolution of 5.1 Å (0.143 threshold
value), and the red curve indicates a resolution of 4.1 Å. By using the more
conservative threshold of 0.5, the cut-off values are 6.5 Å (black curve) and 4.5
Å (red curve).
Zhang et al.
Fig. 5. Correlation coefficients between images of DLPs and the reference.
(a) Correlation coefficients are plotted as a function of particle and batch
number (red, lacy carbon; green, C-flat). There is a bimodal distribution with
a correlation coefficient of !0.14 separating the two clusters. (b) Correlation
coefficients are plotted as a function of image defocus. (c) Correlation coefficients are plotted as a function of approximate particle distance from the
center of hole in the carbon film.
was least noticeable in samples prepared by using C-flat grids,
suggesting that the ice thickness in those samples was more
uniform than in grids prepared with lacy carbon.
Nonicosahedral Averaging. We used the atomic models for VP2
and VP6 built into the x-ray map to interpret the cryo-EM
structure. In the icosahedrally averaged reconstruction of the
DLP, the density corresponding to most aromatic side chains is
clearly visible, whereas other, mostly smaller, side chains appear
in incomplete or displaced density (Figs. 2d and 3).
The 780 VP6 monomers in the outer shell of DLP assume T #
13 icosahedral symmetry. The density map of VP6 therefore
could be improved further by averaging the density of the 13
equivalent monomers that are not icosahedrally related. Bundles
of three generic !-helices, generated with the computer program
MOLEMAN (17, 18) and containing one 8-alanine and two
11-alanine helices, were placed in clearly identifiable density
features of the three VP6 monomers around one of the icosahedral threefold axes. For the placement, we used a density map
filtered at 7-Å resolution and sharpened with a B factor of $350
Å2 (Fig. 2b). The low-pass filter had a cosine-shaped cut-off with
a width of !5 Fourier pixels. Copies of the trimer of helical
bundles then were placed in the four other VP6 trimer densities
containing the remaining 12 equivalent monomers (Fig. 2a).
Approximate matrices transforming the coordinates of these
four trimers into the trimer on the icosahedral threefold axis
PNAS " February 12, 2008 " vol. 105 " no. 6 " 1869
BIOPHYSICS
tion of only 25 Å. Thus, the correlation coefficient is a clear
indicator of a usable particle.
The number of usable particles depends strongly on the batch
number of the virus DLP preparation. Approximately 30% of the
virus particles collected from the first batch fell below the
threshold criterion for inclusion in the reconstruction, whereas
it was !75% in the second batch (Fig. 5a). Data from both
batches were recorded on several days and from different cryo
grids, and properly aligned particles from both batches had
approximately the same average correlation coefficient. Almost
all micrographs contained both usable and discarded particles.
However, virus particles from the first batch were frozen by using
homemade lacy carbon, whereas material from the second batch
was frozen by using commercially available C-flat grids that were
lightly recoated with carbon before use to enhance conductivity.
The dependence on batch number might reflect a difference in
the quality of the particles. It is more likely, however, that the
data from the second batch contained more misaligned particles
because they were added at a different stage in the image
processing, compared with the first batch (see Materials and
Methods).
The correlation coefficient also depends on the defocus used
to collect the data (Fig. 5b). This dependence is not surprising
because the defocus determines the spectral distribution of the
signal in the image. A larger defocus amplifies the signal at low
resolution where the molecular transform of the virus particle is
strongest. Corresponding particles therefore show a higher correlation coefficient than particles imaged with a smaller defocus.
The correlation coefficient also depends to some degree on the
location of the virus particle in the carbon film hole (Fig. 5c).
Particles closer to the edge of a hole tend to have a smaller
correlation coefficient than particles closer to the center. We
measured the ice thickness by burning a small hole into the ice
layer at a tilt of $45° and then observing the hole length at a tilt
of %45°. The measurements showed that the ice thickness close
to the carbon was !1,200 Å, whereas it was only 700 Å near the
center of the hole, similar to the diameter of the DLP. Thicker
ice increases the fraction of inelastically and multiply scattered
electrons, which add to the background in an image and reduce
the signal produced by the remaining singly and elastically
scattered electrons. This reduction in signal lowers the correlation coefficient between particle and reference. Some particles
too close to the hole center also exhibited a slight decrease in
correlation coefficient. These particles might have suffered some
deformation because of the thin ice. The dependence on location
Fig. 6. Comparison of x-ray crystallographic and EM density maps. (a) X-ray crystallographic, icosahedrally averaged 2Fo $ Fc density map at 3.8-Å resolution
and atomic model built into this map (B. McLain, E.S., R.B., and S.C.H., unpublished data). The density corresponds to area 2 in Fig. 2c. (b) Same area as in a but
showing the cryo-EM map. (c) Cryo-EM density in area 3 (Fig. 2c) showing clearly separated "-sheet strands. (d) Stereo image of area 4 (Fig. 2c) showing amino
acid side chains protruding from different !-helices.
were determined by using the computer program LSQMAN (17,
18). The matrices were refined with the computer program IMP
(17, 18) by aligning densities in the vicinity of the trimers of
helical bundles against equivalent density of the VP6 trimer on
the threefold icosahedral symmetry axis. For the refinement, we
used a density map filtered at 4-Å resolution and sharpened with
a B factor of $300 Å2. As before, the low-pass filter had a
cosine-shaped cut-off with a width of !5 Fourier pixels. A full
set of 12 transformation matrices was derived from the 4 initial
matrices by combining the matrix transformations with additional 120° rotations around the threefold icosahedral symmetry
axis. The VP6 densities were averaged by using the 12 matrices
and the computer program AVE (17, 18). For the averaging, an
ovoid mask with radii of 50 and 60 Å in the two orthogonal
directions was defined that generously enveloped the VP6 trimer
on the threefold axis. The refined matrix transformations were
compared with transformations derived from an atomic model
built into the x-ray map (B. McLain, E.S., R.B., and S.C.H.,
unpublished data). Coordinates transformed by the refined
matrices deviated from those transformed by using matrices
based on the atomic model with an rmsd of 0.7 Å. This alignment
error contributed further to the signal attenuation in the final
structure (see below).
The final 13-fold averaged density is shown in Figs. 2e and 6
b–d. It has clear features for most of the larger side chains,
including aromatic, aliphatic, polar, and charged side chains. The
map compares well with an electron density map determined by
x-ray crystallography (B. McLain, E.S., R.B., and S.C.H., unpublished data) at 3.8-Å resolution (Fig. 6a), effectively calibrating the resolution of the 13-fold averaged density. The
resolution also was estimated by using the Fourier shell correlation (FSC) criterion (Fig. 4), suggesting a resolution of !4 Å.
The interpretation of the FSC curve comparing 13-fold averaged
densities of reconstructions using two halves of the data is
complicated by the tight masking imposed during the averaging
procedure (see Materials and Methods), making the resolution
estimate less accurate. The influence of the mask on the FSC was
reduced by smoothing the edges of the mask.
Discussion
The resolution obtained in the present work represents a substantial step forward in the application of cryo-EM. The promise
of achieving a map interpretable by an atomic model has driven
the development of new sample preparation techniques, instrumentation, and image processing methods (13, 19–21). An
estimate of the number of images that might be required to attain
near-atomic resolution was made 12 years ago (22). Subsequent
single-particle reconstructions of hepatitis B virus capsids at 7to 9-Å resolution were adequate to resolve secondary structures
and permitted an approximate trace of the subunit polypeptide
chain (23, 24). Virus particles are particularly suitable for
high-resolution imaging because of their high symmetry, their
generally high stability, and their solubility in aqueous buffers.
Moreover, their high molecular mass gives rise to strong contrast
in the EM, but limitations commonly encountered in cryo-EM
applications, such as beam-induced movement and specimen
charging, greatly reduce image contrast from the values expected
under ideal conditions. Early estimates of the minimum number
of images required to obtain a 3-Å resolution structure ranged
between 1,400 and 12,600 (22, 25), depending on the assumed
Zhang et al.
Materials and Methods
EM. Two batches of rotavirus DLPs were prepared as described (29). DLP stock
solution was diluted to !5 mg/ml, and 2 #l of batch no. 1 was applied to lacy
carbon grids, whereas 2 #l of batch no. 2 was applied to C-flat holey carbon
grids (CF-1/2– 4C; Electron Microscopy Sciences). Images were collected on
Kodak ISO163 film by using an FEI F30 electron microscope at 300 kV, "59,000
magnification, and a Gatan 626 cryo holder. We used an underfocus of 1.1–3.5
#m and an electron dose of 15–20 e$/Å. The spot size was set to 6, and a C2
aperture of 50 #m was used, giving a beam cross-over of !5 nm and divergence of 5 #rad. This results in a spatial coherence width of !600 Å (30),
sufficient to preserve contrast at 3.8-Å resolution and 3.5-#m defocus. The
beam pivot points were centered with the specimen at eucentric height, and
the beam tilt was aligned by aligning the objective rotation center. The
condenser astigmatism was minimized, followed by coma-free alignment.
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Zhang et al.
SEE COMMENTARY
These last two steps were repeated several times until there was no further
improvement. Wherever possible, the beam was placed symmetrically over a
hole in the carbon film to ensure the presence of carbon on all sides of the
illuminated area.
A calibrated magnification of 58,080 was used for contrast transfer correction and image processing by FREALIGN. A more accurate magnification of
56,900 subsequently was determined with a standard (tropomyosin paracrystal) and used to perform nonicosahedral averaging. Comparison with the
structure determined by x-ray crystallography gave a magnification of 56,540,
close to the calibrated value.
Image Processing. We initially selected 386 micrographs by visual inspection
and digitized them using a Zeiss SCAI scanner with a 7-#m step size, giving a
pixel size on the specimen of !1.2 Å. Processing of the data was accelerated
by initial decimation of the images with 2 " 2-pixel averaging. Power spectra
from each micrograph (including both carbon and ice areas) were calculated
to select for micrographs with preserved high-resolution signal. Then, 8,696
and 9,429 particles were selected from images of batch nos. 1 and 2, respectively, by using the computer program SIGNATURE (31). The astigmatic defocus, specimen tilt axis, and tilt angle for each micrograph were determined by
using the computer program CTFTILT (32), giving separate defocus values for
each particle according to its coordinate in the micrograph. The detected
specimen tilt was unintentional and remained &5° for most micrographs.
By using the search function of the computer program FREALIGN (13) with
a 1° angular step, particles collected from batch no. 1 initially were aligned
against a structure of reovirus (33) filtered at 30-Å resolution and scaled to
match the diameter of DLP. This step was followed by 70 cycles of refinement,
converging to a structure of DLP. For the refinement, the density corresponding to the RNA genome was masked in the reference structure to enhance the
signal of the ordered part of the structure. Particles collected from batch no.
2 were added and aligned to the DLP structure by using FREALIGN's search
function. Fifteen additional refinement cycles at the full resolution of 1.2
Å/pixel—followed by randomized parameter search (13), another refinement
cycle, and a final selection of 8,400 particles with a correlation coefficient
(0.14 —resulted in a structure of 5.1-Å resolution before nonicosahedral
averaging. For the randomized parameter search, a coarse search grid with
angular step of 50° was used. This grid search was repeated 50 times for each
particle by using random starting points, leading to different searched orientations in each grid search. The resolution was determined by using the FSC
criterion and calculated between two reconstructions each containing half of the
data and a threshold of 0.143 (34). A negative temperature factor of 450 Å2 (x-ray
notation) was applied, and the structure was filtered at 3.8-Å resolution. The
low-pass filter had a cosine-shaped cut-off with a width of !20 Fourier pixels.
Estimation of Alignment Errors. A reference structure was calculated with half
of the aligned data used to calculate the 5.1-Å icosahedral reconstruction. The
reference was not improved with 13-fold nonicosahedral averaging. The
alignment parameters then were altered by adding small angular perturbations with a uniform distribution of )3° and small positional perturbations of
)3.5 Å. One cycle of refinement was carried out with FREALIGN, which
brought most of the parameters of particles with a high correlation coefficient
(see above) back close to their starting values before perturbation. Some
particles were not refined successfully and ended up with a significantly lower
correlation coefficient, compared with those refined successfully. These particles, and a small number of other particles with large angular deviations
from the starting angles, were not considered further. The standard deviation
between the refined and the starting parameters was calculated and used to
estimate the parameter errors.
ACKNOWLEDGMENTS. This work was supported by National Institutes of
Health Grants GM-62580 (to N.G. and S.C.H.), CA-13202 (to S.C.H.), and AI53174 (to P.R.D. and S.C.H.), an Ellison Medical Foundation New Scholars in
Global Infectious Diseases Award (to P.R.D.), and grants from the Health
Research Council of New Zealand (to R.B.). N.G. and S.C.H. are Howard Hughes
Medical Institute Investigators.
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PNAS " February 12, 2008 " vol. 105 " no. 6 " 1871
BIOPHYSICS
signal-to-noise ratio required in an interpretable reconstruction.
Both estimates assumed physically perfect image formation,
without loss of contrast because of the limitations mentioned
above. Contrast in experimentally observed images usually is
only !10% of the contrast expected in a perfect image (22). In
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of 0.2° and x, y coordinates within 0.2 Å. An angular error of 0.2°
is expected to attenuate the signal in the reconstruction of
a 700-Å particle by a factor of !0.3, whereas a 0.2-Å x, y error
leaves the signal almost unchanged (26). A second limitation
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to record images. This error has been estimated to be !100 Å for
images containing carbon film and !1,000 Å for images without
carbon. The imaged specimen area included in each micrograph
contained !50% carbon, and the virus particles themselves also
contributed significantly to the Thon ring pattern used to
determine the defocus. If we assume a defocus error of 300 Å,
we expect the signal in the reconstruction to be attenuated by a
further factor of 0.4. Other limitations that are not detectable as
easily include magnification variations among images (27) and
unintentional beam tilt. Finally, the data were not corrected for
the curvature of the Ewald sphere, introducing a phase error of
!60° at 3.8-Å resolution (28), which will further attenuate the
signal in the reconstruction by a factor of !0.5. The attenuation
therefore is similar to that expected for a defocus error of half
the particle diameter (350 Å).
The ability to trace the protein backbone in a structure introduces
powerful additional constraints, which have been used in x-ray
crystallography to improve the density map and ultimately the
coordinates (‘‘refinement’’). Now that traceable maps also can be
obtained by using single-particle reconstruction from cryo-EM,
new refinement methods can be considered. For example, a partial
model of a structure could be used to improve the reference used
for particle alignment. A partial model also could be used to
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constraint in a refinement or to derive masks that reduce or remove
noisy parts of the reconstructed image.
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Zhang et al.

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