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- EMDB-16699: Subtomogram average of HIV-1 CA hexamer from capsid-like particle... -

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Entry
Database: EMDB / ID: EMD-16699
TitleSubtomogram average of HIV-1 CA hexamer from capsid-like particles assembled with inositol hexakisphosphate
Map dataSubtomogram average of HIV-1 CA protein hexamer from capsid-like particles assembled with inositol hexakisphosphate
Sample
  • Complex: HIV-1 CA protein capsid-like particles assembled in the presence of inositol hexakisphosphate
Biological speciesHuman immunodeficiency virus 1
Methodsubtomogram averaging / cryo EM / Resolution: 3.9 Å
AuthorsTan A / Briggs JAG / Dick RA
Funding support United States, United Kingdom, Germany, 8 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI147890 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)5U54AI150472-09 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U54 AI170855-01 United States
Medical Research Council (MRC, United Kingdom)MC_UP_1201/16 United Kingdom
Max Planck Society Germany
Other privateRRID:SCR_021727
Other privateRRID:SCR_021740
National Science Foundation (NSF, United States)DMR-1719875 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structural insights into HIV-1 polyanion-dependent capsid lattice formation revealed by single particle cryo-EM.
Authors: Carolyn M Highland / Aaron Tan / Clifton L Ricaña / John A G Briggs / Robert A Dick /
Abstract: The HIV-1 capsid houses the viral genome and interacts extensively with host cell proteins throughout the viral life cycle. It is composed of capsid protein (CA), which assembles into a conical ...The HIV-1 capsid houses the viral genome and interacts extensively with host cell proteins throughout the viral life cycle. It is composed of capsid protein (CA), which assembles into a conical fullerene lattice composed of roughly 200 CA hexamers and 12 CA pentamers. Previous structural analyses of individual CA hexamers and pentamers have provided valuable insight into capsid structure and function, but detailed structural information about these assemblies in the broader context of the capsid lattice is lacking. In this study, we combined cryoelectron tomography and single particle analysis (SPA) cryoelectron microscopy to determine structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also developed a method of liposome scaffold-based in vitro lattice assembly ("lattice templating") that enabled us to directly study the lattice under a wider range of conditions than has previously been possible. Using this approach, we identified a critical role for inositol hexakisphosphate in pentamer formation and determined the structure of the CA lattice bound to the capsid-targeting antiretroviral drug GS-6207 (lenacapavir). Our work reveals key structural details of the mature HIV-1 CA lattice and establishes the combination of lattice templating and SPA as a robust strategy for studying retroviral capsid structure and capsid interactions with host proteins and antiviral compounds.
History
DepositionFeb 15, 2023-
Header (metadata) releaseMay 3, 2023-
Map releaseMay 3, 2023-
UpdateMay 3, 2023-
Current statusMay 3, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16699.png

Downloads & links

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Map

FileDownload / File: emd_16699.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of HIV-1 CA protein hexamer from capsid-like particles assembled with inositol hexakisphosphate
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.38 Å/pix.
x 192 pix.
= 264.768 Å		1.38 Å/pix.
x 192 pix.
= 264.768 Å		1.38 Å/pix.
x 192 pix.
= 264.768 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.379 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.707
Minimum - Maximum-2.1272545 - 3.9188676
Average (Standard dev.)0.0015233356 (±0.26237142)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 264.768 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Subtomogram average of HIV-1 CA protein hexamer from...

Fileemd_16699_half_map_1.map
AnnotationSubtomogram average of HIV-1 CA protein hexamer from capsid-like particles assembled with inositol hexakisphosphate, half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Subtomogram average of HIV-1 CA protein hexamer from...

Fileemd_16699_half_map_2.map
AnnotationSubtomogram average of HIV-1 CA protein hexamer from capsid-like particles assembled with inositol hexakisphosphate, half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : HIV-1 CA protein capsid-like particles assembled in the presence ...

EntireName: HIV-1 CA protein capsid-like particles assembled in the presence of inositol hexakisphosphate
Components
  • Complex: HIV-1 CA protein capsid-like particles assembled in the presence of inositol hexakisphosphate

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Supramolecule #1: HIV-1 CA protein capsid-like particles assembled in the presence ...

SupramoleculeName: HIV-1 CA protein capsid-like particles assembled in the presence of inositol hexakisphosphate
type: complex / ID: 1 / Chimera: Yes / Parent: 0
Source (natural)Organism: Human immunodeficiency virus 1

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 6.2
Component:
ConcentrationFormulaName
50.0 mMMESMES
2.5 mMC6H18O24P6Inositol hexakisphosphate
1.0 mMC9H15O6PTris(2-carboxyethyl)phosphine
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
Details: The grid was glow discharged for 30 seconds at a current of 20 mA.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: subTOM / Number subtomograms used: 89951
ExtractionNumber tomograms: 66 / Number images used: 570742 / Software - Name: MATLAB
Details: HIV-1 CA protein capsid-like particles (CLPs) were segmented using the Ilastik software package. The vertex coordinates of the segmented volumes were used to define an oversampled grid of ...Details: HIV-1 CA protein capsid-like particles (CLPs) were segmented using the Ilastik software package. The vertex coordinates of the segmented volumes were used to define an oversampled grid of points normal to the CLP surface in MATLAB.
Final angle assignmentType: OTHER / Software - Name: subTOM

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