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- PDB-3j4f: Structure of HIV-1 capsid protein by cryo-EM -

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Basic information

Entry
Database: PDB / ID: 3j4f
TitleStructure of HIV-1 capsid protein by cryo-EM
Componentscapsid proteinCapsid
KeywordsVIRAL PROTEIN / HIV-1 capsid / core / all-atom model / MDFF / tubular assembly / hexamer
Function / homology
Function and homology information


viral budding via host ESCRT complex / viral nucleocapsid / host cell cytoplasm / structural molecule activity / virion membrane / RNA binding / zinc ion binding / identical protein binding / cytoplasm
Similarity search - Function
Gag protein p6 / Gag protein p6 / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retrovirus capsid, C-terminal / Retroviral matrix protein ...Gag protein p6 / Gag protein p6 / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retrovirus capsid, C-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile.
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus 1
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 8.6 Å
AuthorsZhao, G. / Perilla, J.R. / Meng, X. / Schulten, K. / Zhang, P.
CitationJournal: Nature / Year: 2013
Title: Mature HIV-1 capsid structure by cryo-electron microscopy and all-atom molecular dynamics.
Authors: Gongpu Zhao / Juan R Perilla / Ernest L Yufenyuy / Xin Meng / Bo Chen / Jiying Ning / Jinwoo Ahn / Angela M Gronenborn / Klaus Schulten / Christopher Aiken / Peijun Zhang /
Abstract: Retroviral capsid proteins are conserved structurally but assemble into different morphologies. The mature human immunodeficiency virus-1 (HIV-1) capsid is best described by a 'fullerene cone' model, ...Retroviral capsid proteins are conserved structurally but assemble into different morphologies. The mature human immunodeficiency virus-1 (HIV-1) capsid is best described by a 'fullerene cone' model, in which hexamers of the capsid protein are linked to form a hexagonal surface lattice that is closed by incorporating 12 capsid-protein pentamers. HIV-1 capsid protein contains an amino-terminal domain (NTD) comprising seven α-helices and a β-hairpin, a carboxy-terminal domain (CTD) comprising four α-helices, and a flexible linker with a 310-helix connecting the two structural domains. Structures of the capsid-protein assembly units have been determined by X-ray crystallography; however, structural information regarding the assembled capsid and the contacts between the assembly units is incomplete. Here we report the cryo-electron microscopy structure of a tubular HIV-1 capsid-protein assembly at 8 Å resolution and the three-dimensional structure of a native HIV-1 core by cryo-electron tomography. The structure of the tubular assembly shows, at the three-fold interface, a three-helix bundle with critical hydrophobic interactions. Mutagenesis studies confirm that hydrophobic residues in the centre of the three-helix bundle are crucial for capsid assembly and stability, and for viral infectivity. The cryo-electron-microscopy structures enable modelling by large-scale molecular dynamics simulation, resulting in all-atom models for the hexamer-of-hexamer and pentamer-of-hexamer elements as well as for the entire capsid. Incorporation of pentamers results in closer trimer contacts and induces acute surface curvature. The complete atomic HIV-1 capsid model provides a platform for further studies of capsid function and for targeted pharmacological intervention.
History
DepositionJul 11, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 24, 2013Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id / _em_software.name

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Structure visualization

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  • Biological unit as representative helical assembly
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
A: capsid protein
B: capsid protein
C: capsid protein
D: capsid protein
E: capsid protein
F: capsid protein


Theoretical massNumber of molelcules
Total (without water)154,2156
Polymers154,2156
Non-polymers00
Water0
1
A: capsid protein
B: capsid protein
C: capsid protein
D: capsid protein
E: capsid protein
F: capsid protein
x 35


Theoretical massNumber of molelcules
Total (without water)5,397,523210
Polymers5,397,523210
Non-polymers00
Water0
TypeNameSymmetry operationNumber
helical symmetry operation35
2


  • Idetical with deposited unit in distinct coordinate
  • helical asymmetric unit
TypeNameSymmetry operationNumber
helical symmetry operation1
3


  • Idetical with deposited unit in distinct coordinate
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
transform to helical frame1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 35 / Rise per n subunits: 7.247 Å / Rotation per n subunits: -31.13 °)

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Components

#1: Protein
capsid protein / Capsid


Mass: 25702.490 Da / Num. of mol.: 6 / Fragment: UNP residues 133-363 / Mutation: A92E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: gag / Plasmid: pET21 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta 2 (DE3) / References: UniProt: Q79791

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: HIV-1 capsid protein / Type: COMPLEX / Details: hexamer / Synonym: HIV CA
Molecular weightValue: 0.025 MDa / Experimental value: NO
Buffer solutionName: 1 M NaCl, 50 mM Tris-HCl / pH: 8 / Details: 1 M NaCl, 50 mM Tris-HCl
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 200 mesh quantifoil R2/1 copper grid
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temp: 95 K / Humidity: 80 %
Details: With 2.5 uL sample on carbon side, add 3 uL dilution buffer (100 mM NaCl, 50 mM Tris, pH 8.0) to back side. Blot 3-5 seconds from back side and plunge into liquid ethane with a homemade plunger.
Method: With 2.5 uL sample on carbon side, add 3 uL dilution buffer (100 mM NaCl, 50 mM Tris, pH 8.0) to back side. Blot 3-5 seconds from back side.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Date: Dec 11, 2010
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 58257 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm / Cs: 2 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: OTHER / Specimen holder type: Polara cartridge / Temperature: 82 K / Temperature (max): 85 K / Temperature (min): 80 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 15 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 27
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1MDFFmodel fitting
2FREALIGN3D reconstruction
CTF correctionDetails: each filament
Helical symmertyAngular rotation/subunit: 31.13 ° / Axial rise/subunit: 7.247 Å / Axial symmetry: C1
3D reconstructionMethod: real space helical reconstruction / Resolution: 8.6 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 3210 / Actual pixel size: 1.09 Å
Details: (Helical Details: The segments were aligned and reconstructed using Frealign. Twofold symmetry was imposed using IHRSR++.)
Symmetry type: HELICAL
Atomic model building
IDProtocolSpaceDetails
1FLEXIBLE FITREALMETHOD--Seven hexamers were docked into density then solvated into 1M NaCl. Secondary structure restraints were applied to helices 1 to 11. MDFF was run for 10 ns with symmetry restraints between CA monomers along the chiral axis. The center hexamer was then extracted. PDB entries 3H47 and 2KOD were the starting structures. REFINEMENT PROTOCOL--flexible
2FLEXIBLE FITREALMETHOD--Seven hexamers were docked into density then solvated into 1M NaCl. Secondary structure restraints were applied to helices 1 to 11. MDFF was run for 10 ns with symmetry restraints between CA monomers along the chiral axis. The center hexamer was then extracted. PDB entries 3H47 and 2KOD were the starting structures. REFINEMENT PROTOCOL--flexible
Atomic model building
IDPDB-ID 3D fitting-ID
12KOD

2kod

1
23H47

3h47

2
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms10799 0 0 0 10799

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