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Basic information

Entry
Database: PDB / ID: 4d1k
TitleCryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly.
ComponentsGAG PROTEINHIV-1 protease
KeywordsVIRAL PROTEIN / CAPSID / SP1 / HELICAL RECONSTRUCTION
Function / homologygag protein p24 N-terminal domain / viral process / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / viral capsid / Retrovirus capsid, C-terminal / Retrovirus capsid, N-terminal / Gag protein
Function and homology information
Biological speciesHUMAN IMMUNODEFICIENCY VIRUS 1
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 9.4 Å
AuthorsBharat, T.A.M. / Castillo-Menendez, L.R. / Hagen, W.J.H. / Lux, V. / Igonet, S. / Schorb, M. / Schur, F.K.M. / Krauesslich, H.G. / Briggs, J.A.G.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly.
Authors: Tanmay A M Bharat / Luis R Castillo Menendez / Wim J H Hagen / Vanda Lux / Sebastien Igonet / Martin Schorb / Florian K M Schur / Hans-Georg Kräusslich / John A G Briggs /
Abstract: The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and ...The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny immature virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within immature virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-Å resolution structure of an immature virus-like tubular array assembled from a Gag-derived protein of the related retrovirus Mason-Pfizer monkey virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.
History
DepositionMay 2, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 4, 2014Provider: repository / Type: Initial release
Revision 1.1Jun 25, 2014Group: Database references
Revision 1.2Aug 23, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: GAG PROTEIN
B: GAG PROTEIN
C: GAG PROTEIN
D: GAG PROTEIN
E: GAG PROTEIN
F: GAG PROTEIN


Theoretical massNumber of molelcules
Total (without water)146,5326
Polymers146,5326
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS

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Components

#1: Protein
GAG PROTEIN / HIV-1 protease


Mass: 24421.988 Da / Num. of mol.: 6 / Fragment: CAPSID, RESIDUES 1-219 / Mutation: YES
Source method: isolated from a genetically manipulated source
Details: 10NM PROTEIN-A CONJUGATED GOLD PARTICLES WERE ADDED TO THE SAMPLE. PROTEIN CONSTRUCT CONSISTS OF CA TO NC DOMAINS WITH MUTATION IN POSITION Y169.
Source: (gene. exp.) HUMAN IMMUNODEFICIENCY VIRUS 1 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q5D0H3
Sequence detailsTHE PROTEIN DEPOSITED IS A MUTANT AT POSITION Y169.

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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 CANC Y169 MUTANT PROTEIN ASSEMBLED WITH 73MER DNA INTO TUBULAR PARTICLES
Type: COMPLEX
Buffer solutionName: 30 MM MES, 1 MM EDTA, 1 MM DTT, 0.5 M NACL / pH: 6 / Details: 30 MM MES, 1 MM EDTA, 1 MM DTT, 0.5 M NACL
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 100, INSTRUMENT- HOMEMADE PLUNGER,

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Dec 25, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 23 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 82
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1MDFFmodel fitting
2UCSF Chimeramodel fitting
3AV33D reconstruction
4SPIDER3D reconstruction
3D reconstructionResolution: 9.4 Å / Nominal pixel size: 1.5 Å / Actual pixel size: 1.53 Å
Details: ATOMIC MODELS WERE FITTED USING MOLECULAR MOLECULAR DYNAMICS FLEXIBLE FITTING (MDFF). THE CA-NTD ATOMIC MODEL PDB ID 2JPR AND THE CA-CTD ATOMIC MODEL PDB ID 4COP WERE USED AS STARTING MODELS. ...Details: ATOMIC MODELS WERE FITTED USING MOLECULAR MOLECULAR DYNAMICS FLEXIBLE FITTING (MDFF). THE CA-NTD ATOMIC MODEL PDB ID 2JPR AND THE CA-CTD ATOMIC MODEL PDB ID 4COP WERE USED AS STARTING MODELS. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2638. (DEPOSITION ID: 12470).
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: METHOD--FLEXIBLE REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 4COP

4cop

RefinementHighest resolution: 9.4 Å
Refinement stepCycle: LAST / Highest resolution: 9.4 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3942 0 0 0 3942

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