Supplement

www.sciencemag.org/cgi/content/full/science.aaf9620/DC1
Supplementary Materials for
An atomic model of HIV-1 capsid-SP1 reveals structures regulating
assembly and maturation
Florian K. M. Schur, Martin Obr, Wim J. H. Hagen, William Wan, Arjen J. Jakobi,
Joanna M. Kirkpatrick, Carsten Sachse, Hans-Georg Kräusslich, John A. G. Briggs*
*Corresponding author. Email: john.briggs@embl.de
Published 14 July 2016 on Science First Release
DOI: 10.1126/science.aaf9620
This PDF file includes:
Materials and Methods
Figs. S1 to S5
Tables S1 to S2
Full Reference List
Captions for Movies S1 and S2
Other Supplementary Material for this manuscript includes the following:
(available at www.sciencemag.org/cgi/content/full/science.aaf9620/DC1)
Movies S1 and S2
Materials and Methods
HIV-1 purification and Virus-like particle assembly
HEK293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with
10% fetal calf serum (FCS; Biochrom), penicillin (100 IU/mL), streptomycin (100
µg/mL) and 4 mM glutamine. For virus production, cells were transfected with the
proviral plasmid pNL4-3 (PR-) using calcium phosphate following standard procedures.
At 44 h post transfection, tissue culture supernatant was harvested and filtered through
0.45 µm nitrocellulose filters. Virus was enriched by ultracentrifugation through a 20%
(w/w) sucrose cushion and further purified by centrifugation through an iodixanol density
gradient (34). Concentrated virus samples were resuspended in phosphate buffered saline
(PBS), treated with 1% paraformaldehyde for 1 h on ice and stored in aliquots at -80°C.
Purity of samples and Gag maturation state were verified by SDS-PAGE followed by
silver staining and immunoblotting.
ΔMACANCSP2 protein was expressed in E. coli cells and purified as described
previously (11). In brief, the cell lysate was precipitated by ammonium sulphate (25%
saturation), and further purified using a combination of anion and cation exchange
chromatography. Homogenously pure ΔMACANCSP2 was transferred into a storage
buffer (50 mM Hepes, pH 7.5, 500 mM NaCl, 10% Glycerol, 10 µM ZnCl2) and
concentrated to 4 mg/ml.
In order to assemble ΔMACANCSP2 VLPs, protein stock was dialyzed against assembly
buffer (50 mM Hepes, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM TCEP) in the
presence of nucleic acid (single-stranded DNA 73mer oligonucleotide, 1:10 molar ratio
oligonucleotide:protein) for 16h at 4°C. In vitro assembled particles were harvested by
centrifugation (10 000g, 5 min) and resuspended in fresh assembly buffer. Additionally,
particles for the binding analysis of the maturation inhibitor BVM were assembled in
presence of the compound (100 µg/ml), and washed in 20% methanol prior to
resuspension. Inhibition of CA-SP1 cleavage was validated by incubating 500 nM
ΔMACANCSP2 VLPs, assembled in the presence or absence of 100µg/ml BVM, with
100 nM recombinant HIV-1 PR in PR assay buffer (50 mM MES, pH 6.5, 150 mM NaCl,
1 mM EDTA, 1mM TCEP, 1mg/ml BSA) for 1h at 25°C. Semi-quantitative western blots
were performed with polyclonal sheep antiserum against CA and secondary antibodies
coupled to Alexa fluorescent dyes for detection with an Odyssey Infrared Imaging
System (Li-Cor Biosciences, Lincoln, NE). Band intensities were quantitated using
ImageJ to determine relative accumulation of CA-SP1 cleavage products (Fig. S5).
Approximate quantification of the amount of BVM incorporated into VLPs was
performed by UPLC-MS-MS (Waters Q-Tof Premier Mass Spectrometer). Protein and
nucleic acid from the BVM containing sample was denatured by a methanol-acetonitrile
mixture (80:20) and separated by retention in a centrifugal filter unit (molecular weight
cut-off 30 kDa). The flow-through was collected, dried in a SpeedVac concentrator at
37°C and reconstituted in UPLC sample buffer (20 mM ammonium formate, pH 8.0,
20% acetonitrile, 5% methanol, 1% DMSO). The reconstituted sample was applied to a
2
C18 reverse phase column and eluted by a linear gradient of 20 – 95% acetonitrile in 20
mM ammonium formate, pH 8.0. The eluate was analyzed by electrospray ionization in
negative ion mode with tandem MS/MS detection (Q-TOF). The integral area of the
583.40 Da BVM monoisotopic peak was used for quantification, compared to a
calibration curve prepared by mixing BVM at known concentrations with pre-assembled
ΔMACANC VLPs. In data from four independent experiments we observed a molar ratio
of 2.6 BVM : 6 ΔMACANCSP2 (range 0.9-4.1) but this may partly reflect non-specific
binding.
Cryo-electron tomography
Degassed 2/1-3C or 2/2-3C C-flat grids were glow discharged for 30 seconds at 20 mA.
VLP or virus solution was diluted with 10nm colloid gold in either PBS or VLP sample
buffer. Then 2 µl of the solution was applied to grids and plunge frozen in liquid ethane.
Grids were stored under liquid nitrogen conditions until imaging.
Data acquisition and image processing was performed identically for all three datasets
(untreated ΔMACANCSP2, ΔMACANCSP2+BVM and immature HIV-1 (D25A) virus
particles) unless otherwise stated (see also Table S1).
All imaging was done on a FEI Titan Krios transmission electron microscope operated at
300 keV, through a Gatan Quantum 967 LS energy filter using a slit width of 20 eV, onto
a Gatan K2xp direct detector using SerialEM software (35). For navigation and search
purposes, low-magnification montages were acquired. Prior to data acquisition, a full K2
gain reference was acquired and the Quantum energy filter was fully tuned. FEI AutoCTF
software (part of the FEI Volta phase plate package) (36) was used for microscope
tuning. The nominal magnification for data collection was 105,000x, giving a calibrated
4K pixel size of 1.35 Å. The tilt range was from 0 to 60° and -60° in 3° steps, in a newlydeveloped dose-symetric tilt scheme (0, +3, -3, -6, +6, +9, -9, -12, etc.), the
implementation of which is described in (12). Tilt images were acquired as 8K x 8K
super-resolution movies of 6-10 frames with a set dose rate of 1.5-8 e/Å2/sec. Tilt series
were collected at a range of nominal defoci between -1.5 and -5.0 µm and a target total
dose of 90 to 150 e/Å2.
Image processing
K2Align software, which uses the MotionCorr algorithm (14), was used to align frames
and Fourier crop the aligned images to 4K x 4K, minimizing aliasing effects. Defocus
determination was performed using CTFFIND4 (37) or by fitting theoretical CTF-curves
to radially-averaged powerspectra using MATLAB (MathWorks) scripts as described
previously (38). CTF-estimation was done for each tilt individually. Each image was lowpass filtered according to the cumulative electron dose (exposure filtering). Exposure
filters were calculated using the exposure-dependent amplitude attenuation function and
critical exposure constants as previously determined (15). For a small number of tiltseries with higher cumulative electron dose, high-tilts were removed prior to further
3
processing.
Exposure-filtered images were then used for tomogram reconstruction in the IMOD
software package (39). CTF-correction was performed by the “ctfphaseflip”-program
implemented in IMOD (40). CTF-corrected unbinned tomograms were reconstructed and
subsequently 2x (2.7 Å/px), 4x (8.1 Å/px) and 8x (10.8 Å/px) binned using anti-aliasing.
VLPs and virus particles were identified in the 8x-binned tomograms using the Amira
visualization software (FEI Visualization Sciences group) and for each particle the center
and radius was determined using the electron microscopy toolbox (41). Tomograms for
untreated ΔMACANCSP2, ΔMACANCSP2+BVM and protease defective HIV-1
(D25A) contained 285 VLPs, 383 VLPs and 484 viruses, respectively. All three datasets
were processed independently.
Sub-volumes with a size of (389)3 Å were extracted from 8x binned tomograms on the
surface of each particle according to the determined radius. Subtomogram averaging was
performed using scripts derived from the AV3 (42), TOM (43) and Dynamo packages
(44). Initial angles were assigned according to the geometry of a sphere. A starting
reference generated from a preliminary dataset containing density for the CA-SP1-NC
region and filtered to 32 Å was used for both untreated ΔMACANCSP2 and
ΔMACANCSP2+BVM datasets. A starting reference for the immature HIV-1 D25A
dataset was generated from a single tomogram. 6-fold symmetry inherent in the structures
was applied throughout processing.
For the VLP datasets two rounds of alignment and averaging were performed.
Subsequently, sub-volumes that had converged onto the same position or that contained
no protein density corresponding to the CA-SP1 layer were removed using a “sub-volume
to sub-volume distance” and cross-correlation threshold, respectively. The remaining
sub-volumes were aligned for one (untreated ΔMACANCSP2) or two more
(ΔMACANCSP2+BVM) iterations.
For the HIV-1 D25A virus dataset four iterations of alignment and averaging were
performed to determine the initial orientations and positions of the sub-volumes on the
lattice. After the first three rounds, sub-volumes that converged onto the same position of
the Gag lattice were removed using a “sub-volume to sub-volume distance”. After
another iteration sub-volumes that contained no Gag protein density were removed
according to their lower cross-correlation values.
Sub-volumes of a size of (389)3 Å were then extracted from 4x-binned tomograms at
positions determined in the 8x-binned alignments and averages were generated by
aligning the sub-volumes according to angles determined in the 8x-binned alignments.
The datasets were aligned for three iterations, with a progressive reduction in angular
search. During alignment, a low-pass filter was applied at 26 Å resolution for the
untreated ΔMACANCSP2 dataset and 32 Å for the ΔMACANCSP2+BVM and HIV-1
(D25A) virus datasets. At the end of the 4x-binned alignments a “sub-volume to subvolume distance” threshold was again applied. The remaining sub-volumes with a box
size of (346)3 Å were extracted at their aligned positions from 2x-binned tomograms. At
this stage, the dataset was split into even/odd half datasets and from this stage on,
even/odd datasets were treated absolutely independently. Sub-volumes with mean grey
4
values further than ±1x standard deviation from the dataset mean (e.g. containing parts of
gold particles) were removed.
For the even/odd datasets independent references were generated by averaging their
respective sub-volumes and filtered to 17 Å. After two more rounds of alignment with
sequentially reduced angular searches, sub-volumes of size (259)3 Å were finally
extracted from unbinned tomograms. Independent references were generated from subvolumes of each respective dataset. Low-pass filters were set to 8.1 Å and sub-volumes
were aligned two more rounds using only 6x1 degree angular sampling cones (out of
plane and in plane). Final averages were generated from 265,506/263,910;
386,040/386,598; and 301,302/301,920 asymmetric units in the even/odd halfsets of the
untreated ΔMACANCSP2, ΔMACANCSP2+BVM and immature HIV-1 (D25A) virus
data, respectively.
The final averages were multiplied with a Gaussian-filtered cylindrical mask and
resolution was determined by mask-corrected Fourier-shell correlation (45). The half
maps were averaged and reweighted by division by their summed CTFs. Finally the
untreated ΔMACANCSP2, ΔMACANCSP2+BVM and HIV-1 (D25A) virus maps were
sharpened with an empirically determined B-factor of -490, -350 or -380 A2, respectively
and filtered to their measured resolutions (46). The structures displayed α-helices
showing helical pitches and clear densities for aromatic, basic and even smaller
hydrophobic side chains consistent with the measured resolutions.
To calculate the difference maps shown in Fig. S2G-I, the amplitude spectra of the
respective maps were scaled, the grey-values of the maps were normalized between 20
and 6 Å, the maps filtered to 6 Å resolution, and a high-pass filter applied at 130 Å to
remove any low-frequency gradients. They were then subtracted from each other to
generate the difference maps.
EM-densities were visualized in Chimera (47), Coot (48) and Pymol (49).
Improvement in resolution
The following differences in data acquisition and image processing contributed to the
increase in resolution from ~8 Å in our previously published structure of immature HIV1 (6) to ~4 Å for the structures presented here. We have collected data using a Gatan
Quantum 967 LS energy filter with Gatan K2xp direct detector, compared to a Gatan
GIF2002 energy filter with CCD camera. The direct electron detector gives significantly
improved detective quantum efficiency, and therefore better image quality. The data was
collected at 300kV instead of 200kV. The larger camera field of view allowed more
efficient collection of a larger dataset. Movie-mode processing was applied to
compensate for beam-induced motion (14). Improved image quality allowed per-tilt
defocus-determination. Use of an optimized dose-symmetric tilt scheme for data
collection improved data quality by redistributing dose-dependent sample damage to the
higher-tilts, thereby improving high-resolution information transfer and minimizing the
impact of does-dependent sample distortions (12). This allowed application of a higher
5
electron dose. The introduction of exposure filtering (15) further improved the signal to
noise ratio in reconstructed tomograms.
Atomic model building and refinement
All refinement was done in the 3.9 Å resolution ΔMACANCSP2+BVM map. In order to
obtain a starting model for coordinate refinement, the NMR model of the CA-NTD
(PDB 1L6N, chain 1, residues 148-279) (16) and the crystal structure of CA-CTD (PDB
3DS2, one monomer, residues 280-353) (19) were rigid body docked into the EM-density
of one CA-monomer using the “Fit in map” option in Chimera (47). A starting model for
the CA-SP1 region was obtained by extracting residues 354-371 from a solution structure
of a CA-SP1 peptide (PDB 1U57) (50), in which residues 351-357 are disordered, and
358-380 form a helix. Chain breaks were joined in Coot (48) and residue Ala301 in PDB
3DS2 was restored to Tyr301 as in the wild-type sequence.
To account for all possible monomer-monomer interactions in CA, a map segment
corresponding to six CA monomers was extracted from the EM density using a mask
extending 3 Å outwards from the center of the rigid-body-fitted model coordinates. We
then performed automated real-space coordinate refinement against the EM density using
a real-space refinement workflow (51, 52) based on CCP4 (53) and cctbx/ PHENIX (54,
55) modules, which was iterated with manual model building in Coot (48) until
convergence.
Briefly, the workflow is as follows. Map segment and model were centered in a cubic
box of P1 symmetry with a cell edge of 324 Å to allow uniform grid sampling of model
and experimental maps at the experimental pixel size. The Cyclophilin-A binding loop
region (residues 216-232) displayed weak density suggestive of multiple conformations.
A single conformation with maximum real-space correlation was obtained after densityguided model deformation (morphing). This conformation was restrained with a weak
harmonic potential in subsequent refinement cycles to scale the global refinement
weights to the poor local resolution in this area. Individual isotropic atomic displacement
parameters (ADPs) were set to 60 Å2 at the beginning of refinement. Real-space
coordinate and ADP refinement was then performed using gradient-driven minimization
of a combined map and restraint target as implemented in PHENIX (54). Noncrystallographic symmetry restraints and secondary structure restraints were applied
throughout. Secondary structure restraints were interrogated and updated during each
refinement iteration based on CaBLAM (56) analysis. Weights on density and geometry
restraints were optimized during each refinement cycle. Each round of model
optimization was evaluated by computing the real-space cross-correlation (RSCC)
between experimental map and a model map simulated by calculating B-factor-weighted
structure factors from the model coordinates. Electron atomic form factors (57) were used
in computation of structure factors. At the end of each iteration, the central monomer of
this assembly was used for generation of the symmetrized model of the biological
assembly as the starting point for the next iteration. The disulfide bond between Cys330
and Cys350 in the CA-CTD was not modeled, but the EM-density suggests it may be
partially occupied.
6
For cross-validation of the final model, model bias was removed by random coordinate
displacement up to a maximum of 0.4 Å, followed by 5 cycles of real-space refinement
against one of the half map reconstructions (work map) using the same refinement
parameters as determined above. We tested for model overfitting by computing the FSC
between the model and the work map (FSCwork) or model and the second half map not
used for coordinate refinement (FSCtest) (Fig. S3A). Significant deviations between both
curves would be a sign of overfitting. The quality of the final model was validated using
MOLPROBITY (58) and was found to range in the top percentile for the corresponding
resolution range.
Comparison of the EM densities of the three samples by FSC (Fig. S2B), by difference
mapping (Fig S2D-I), and by visual inspection (Fig. S3C) suggested that there were no
substantial differences in protein structure. To identify any small local differences in
protein structure we refined the ΔMACANCSP2+BVM model into the EM densities of
the untreated ΔMACANCSP2 and immature HIV-1 (D25A) samples as above to generate
structural models for those samples. We then calculated the root mean square deviations
(RMSDs) in atom position between the ΔMACANCSP2+BVM model and these new
models. C-alpha RMSDs were 0.49 Å and 0.48 Å, and all-atom RMSDs were 1.14 Å and
1.21 Å respectively. Residue-by-residue all-atom RMSDs were below 1 Å for all residues
except six in untreated ΔMACANCSP2, and three in immature HIV-1 (D25A), which are
located in intrinsically flexible regions or can be attributed to resolution variation
between the maps. We conclude that at the determined resolution there are no substantial
differences in protein structure between the three samples.
7
A
B
Protease and Maturation inhibitors
SP1
55kDa Gag
MA
CA
MA
CA
SP2
NC
p6
Maturation
SP1
SP2
NC
p6
Budding
SP2
NC
p6
SP1
MA
Maturation inhibitors
Extracellular
CA
Cytoplasm
Assembly
NC
CA
SP2
SP1
CA
NC
C
SP1
MA
HIV-1
CA-NTD
1
CA-CTD
132
D
363
H1
133
SP2
p6
NC
H2
377
432
448
H3
500
192
PIVQNLQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG
CypA-BL
H4
193
H6
H5
252
GHQAAMQMLK ETINEEAAEW DRLHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTH
310
H7
253
H8
H9
312
NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE
H9
313
H10
H11
CA-SP1 Helix
372
VKNWMTETLL VQNANPDCKT ILKALGPGAT LEEMMTACQG VGGPGHKARV LAEAMSQVTN
373
PATIM
377
Fig. S1.
Schematic representation of the late stage of the HIV-1 lifecycle and the structure of
Gag. (A) The late stage of the virus lifecycle consists of assembly, budding and
maturation. Gag oligomerization starts in the cytoplasm and is driven by protein contacts
mediated by the CA domains and genome-NC interactions. Gag oligomers then shuttle to
the plasma membrane and form hexameric lattices. Upon budding the virus forms
roughly spherical particles of varying dimensions with the Gag lattice underlining the
viral membrane in the shape of a truncated sphere with irregular defects. During
maturation, proteolytic cleavage by the viral protease causes the separation of the
individual Gag domains and rearrangement of the virus structure into the mature
8
infectious form. Maturation can be blocked by PR inhibitors and MIs. (B) The ordered
cascade of cleavage at the five cleavage sites in Gag. The final cleavage, between CA and
SP1, is blocked by the maturation inhibitors. (C) Schematic representation of the HIV-1
Gag domain composition. The extent of the ΔMACANCSP2 construct is shown with
black rectangles. (D) Sequence of HIV-1 CA-SP1. The positions of α-helices are shown
as cyan and yellow bars. The Cyclophilin A binding loop is indicated with a cyan
ellipsoid. Regions for which published structural data is lacking are dashed. Triangles
denote protease cleavage sites. The final proteolytic cleavage during maturation occurs
between L363 and A364.
9
Fig. S2
Comparisons of cryo-electron tomography and subtomogram averaging
reconstructions of the immature HIV-1 CA-SP1 lattice. (A-B) Fourier Shell
correlations (FSC) between independent half datasets for untreated ΔMACANCSP2
10
(red), ΔMACANCSP2+BVM (blue) and immature HIV-1 (D25A) (green). Measured
resolutions are marked in the figure. (B) FSCs between ΔMACANCSP2+BVM and
untreated ΔMACANCSP2 structures (green), between ΔMACANCSP2+BVM and
immature HIV-1 (D25A) solved in this study (light blue) and between
ΔMACANCSP2+BVM and the previously solved 8.8 Å structure of immature HIV-1 CA
(EMD-2706) (purple). (C) Isosurface comparison of the CA-SP1 reconstructions from
the VLPs and the PR defective (D25A) virus obtained in this study to the previously
solved CA-SP1 structure from within PR-inhibited immature HIV-1 viruses (emd-2706).
When filtered to the comparable resolution of 8.8 Å, all CA-SP1 structures are the same.
(D-F) Left and right panels show radial orthoslices through the final unsharpened CASP1 averages of untreated ΔMACANCSP2, ΔMACANCSP2 + BVM and protease
defective HIV-1 (D25A) filtered between 130 Å and 6 Å. Density is white. Scale bars are
25 Å. (G-I) Left hand panels show difference maps calculated by subtracting the
structures in the left panels of D-F from those in the right panels of D-F (13). Right hand
panels shows the difference maps in three-dimensions in red. The isosurface of the
untreated ΔMACANCSP2 structure is shown in grey for orientation purposes.
Overall, no clear differences in the CA-SP1 structure are observed (see also (13)).
Differences are seen at the position of the density coordinated by the lysine rings
(arrowhead) (K290 and K359, Fig 2F) and in the center of the six-helix bundle (arrow).
The density coordinated by the lysine rings is present in all samples but shows
differences in the three maps (arrowheads). This density appears more compact upon
addition of BVM to the VLPs, giving rise to the adjacent positive and negative densities
in G. This density appears much weaker in the PR defective HIV-1 sample than in the
VLPs, giving rise to a positive signal at this position in the difference maps in H and I.
Additional density is seen in the center of the CA-SP1 6-helix bundle in the +BVM
sample (arrow), that gives rise to a positive signal at this position in the difference maps
in G and I.
The density located at the center of the lysine rings is presumably contributed by
negatively charged small molecules or ions coordinated by these rings. This density
cannot be explained as an experimental artifact because it is seen at all equivalent sixhelix bundles within each solved structure, not just at the central one where six-fold
symmetry is applied, and because it is seen at this position in our previous 8.8 Å structure
(6), and in the 8 Å resolution structure of immature-like tubular arrays solved by helical
reconstruction (59). We hypothesize that the molecules coordinated at this position differ
between the immature virus and the in vitro assembled VLPs leading to the weaker
density in the virus.
We attribute the additional density at the center of the six-helix bundle in the +BVM
sample to BVM in its binding site. This density is seen at all equivalent six-helix bundles
within the solved structure. An asymmetric molecule located directly on the 6-fold access
will be smeared-out by the application of 6-fold symmetry, and the orientation of the
molecule therefore cannot be defined.
11
Fig. S3
Refinement of the immature HIV-1 CA-SP1 model. (A) FSCs between the refined
model and the half map used for refinement (FSCwork, blue), between the refined model
and the other half map (FSCtest, green) and between the refined model and the full map
(FSCref, red). The similarity of the FSCwork and FSCtest curves indicate that the model is
not over-refined. (B) Distribution of B-factors in the refined model. Flexible regions, in
particular the Cyclophilin A binding loop (residues 216-232), have higher B-factors (red),
correlating with lower local map resolution due to increased structural flexibility. (C)
Representative EM-densities for immature HIV-1, untreated ΔMACANCSP2, and
ΔMACANCSP2 + BVM, superimposed with the model refined against the 3.9 Å
ΔMACANCSP2 + BVM map. The model is consistent with all three structures,
illustrating the similarity in the protein structure in the three samples. The densities for
ΔMACANCSP2 + BVM are shown at different isosurface thresholds to illustrate the high
quality of the density. The α-helices show helical pitches and clear densities for aromatic,
basic and even smaller hydrophobic side chains.
12
Fig. S4
Contacts within the CA-NTD layer, and at the CA-CTD dimer interface. (A) The
CA-NTD layer viewed from outside the particle. One CA-NTD monomer is highlighted
and the refined side chain positions are shown. The general arrangement of the CA-NTD
is consistent with previous models (6). α-helices are annotated and symmetry axes are
marked. Colored rectangles indicate interfaces shown in the insets. The inter-hexamer
interactions are formed by extensive two-fold and three-fold interfaces and appear to be
stabilized by a salt bridge between residues E207/E208 in helix 4 (mutation of which
interferes with assembly (22)) and R150 in helix 1 of the three-fold related capsid
monomer. Stabilizing contacts around the hexameric ring involve R214 (helix 4), which
projects towards N185 and T186 (helix 3) (B) The CA-CTD layer viewed as in (A). The
highly conserved hydrophobic residues in helix 9 (W316 and M317) form the dimeric
CA-CTD interface. These are the same residues that form the dimeric CA-CTD interface
in the mature virus.
13
A
Untreated
+BVM
CA-SP1
CA
B
**
% CA-SP1 accumulation
80
70
60
50
untreated
+BVM
Fig. S5
In vitro cleavage of ΔMACANCSP2 VLPs. (A) Western blot analysis of untreated
ΔMACANCSP2 and ΔMACANCSP2 + BVM VLP cleavage by recombinant HIV-1 PR
(13). (B) CA and CA-SP1 bands were quantified using ImageJ and accumulation of CASP1 cleavage intermediates were calculated as ratio of band intensities: CA-SP1 / (CASP1+CA). ** denotes p<0.01, error bars denote standard deviation, N=3. BVM leads to a
delay in cleavage between CA and CA-SP1.
14
Table S1.
Summary of Data acquisition and Image processing statistics
Sample
Acquisition settings
HIV-1 ΔMACANCSP2 VLPs
HIV-1 ΔMACANCSP2 VLPs
+ 100 µg/ml Bevirimat
Immature HIV-1 (D25A)
virus
Microscope
FEI Titan Krios
FEI Titan Krios
FEI Titan Krios
Voltage (keV)
300
300
300
Detector
Gatan Quantum K2
Gatan Quantum K2
Gatan Quantum K2
Energy-filter
Yes
Yes
Yes
Slit width (eV)
20
20
20
Super-resolution mode
Yes
Yes
Yes
Å/pixel
1.35
1.35
1.35
Defocus range (microns)
-1.5 to -4.5
-1.5 to -5.0
-1.5 to 5.0
Defocus step (microns)
0.25
0.25
0.25
Acquisition scheme
-60/60°, 3°, Serial EM
-60/60°, 3°, Serial EM
-60/60°, 3°, Serial EM
Total Dose (electrons/Å )
~90 - 270
~120 - 145
~120-221
Dose rate
2
(electrons/Å /sec)
~3 - 8
~3 - 3.8
~1.5 – 5.5
Frame number
6 – 10
8 – 10
10 – 12
Tomogram number
93
43
74
VLPs/Viruses
285
383
484
Asymmetric units Set A
265,506
386,040
301,302
Asymmetric units Set B
263,910
386,598
301,920
Final resolution (0.143
FSC) in Å
4.5
3.9
4.2
2
Processing settings
15
Table S2.
Summary of model refinement statistics
Refinement
HIV-1 ΔMACANCSP2
Residues 143-373
Number of residues
223
All-atom clash score
2.34
Favored rotamers
97.61%
Ramachandran outliers
0
Ramachandran favored
90.24%
C-beta deviations
0
Rmsd (angles, degree)
0.72
Rmsd (bonds ,Å)
0.003
16
Movie S1
A 3D-visualization of the HIV-1 CA-SP1 structure at 3.9 Å resolution.
Movie S2
A guided tour through the structure highlighting structural features presented in this
study.
17
References and Notes
1. W. I. Sundquist, H. G. Kräusslich, HIV-1 assembly, budding, and maturation. Cold
Spring Harb. Perspect. Med. 2, a006924 (2012). Medline
doi:10.1101/cshperspect.a015420
2. B. Müller, M. Anders, H. Akiyama, S. Welsch, B. Glass, K. Nikovics, F. Clavel,
H.-M. Tervo, O. T. Keppler, H.-G. Kräusslich, HIV-1 Gag processing
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