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- PDB-6t61: A model of the EIAV CA-SP hexamer (C2) from Gag-deltaMA tubes ass... -

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Basic information

Entry
Database: PDB / ID: 6t61
TitleA model of the EIAV CA-SP hexamer (C2) from Gag-deltaMA tubes assembled at pH8
ComponentsGag polyproteinGroup-specific antigen
KeywordsVIRAL PROTEIN / Retrovirus / lentivirus / Equine infectious anemia virus / EIAV / Gag / capsid / IP6 / phytic acid / inositolhexakiphosphate
Function / homology
Function and homology information


viral budding via host ESCRT complex / viral nucleocapsid / structural constituent of virion / nucleic acid binding / zinc ion binding
Similarity search - Function
Gag protein p15 / Gag protein p15 / 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 ...Gag protein p15 / Gag protein p15 / 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 speciesEquine infectious anemia virus
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 3.7 Å
AuthorsDick, R.A. / Xu, C. / Morado, D.R. / Kravchuk, V. / Ricana, C.L. / Lyddon, T.D. / Broad, A.M. / Feathers, J.R. / Johnson, M.C. / Vogt, V.M. ...Dick, R.A. / Xu, C. / Morado, D.R. / Kravchuk, V. / Ricana, C.L. / Lyddon, T.D. / Broad, A.M. / Feathers, J.R. / Johnson, M.C. / Vogt, V.M. / Perilla, J.R. / Briggs, J.A.G. / Schur, F.K.M.
Funding support Austria, United States, United Kingdom, Germany, 10items
OrganizationGrant numberCountry
Austrian Science FundP31445 Austria
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01-AI147890 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM107013 United States
National Science Foundation (United States)1659534 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P30-GM110758 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P50AI150481 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI142263 United States
European Research CouncilERC-2014-CoG 648432 - MEMBRANEFUSION United Kingdom
Medical Research Council (United Kingdom)MC_UP_1201/16 United Kingdom
German Research FoundationBR 3635/2-1 Germany
CitationJournal: PLoS Pathog / Year: 2020
Title: Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly.
Authors: Robert A Dick / Chaoyi Xu / Dustin R Morado / Vladyslav Kravchuk / Clifton L Ricana / Terri D Lyddon / Arianna M Broad / J Ryan Feathers / Marc C Johnson / Volker M Vogt / Juan R Perilla / ...Authors: Robert A Dick / Chaoyi Xu / Dustin R Morado / Vladyslav Kravchuk / Clifton L Ricana / Terri D Lyddon / Arianna M Broad / J Ryan Feathers / Marc C Johnson / Volker M Vogt / Juan R Perilla / John A G Briggs / Florian K M Schur /
Abstract: Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that ...Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.
History
DepositionOct 17, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 15, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jul 29, 2020Group: Data collection / Category: em_imaging_optics
Item: _em_imaging_optics.chr_aberration_corrector / _em_imaging_optics.phase_plate / _em_imaging_optics.sph_aberration_corrector
Revision 1.3Mar 30, 2022Group: Author supporting evidence / Database references / Category: database_2 / pdbx_audit_support
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Gag polyprotein
B: Gag polyprotein
C: Gag polyprotein
F: Gag polyprotein
D: Gag polyprotein
H: Gag polyprotein
I: Gag polyprotein
E: Gag polyprotein
G: Gag polyprotein
J: Gag polyprotein
K: Gag polyprotein
L: Gag polyprotein
O: Gag polyprotein
M: Gag polyprotein
Q: Gag polyprotein
R: Gag polyprotein
N: Gag polyprotein
P: Gag polyprotein


Theoretical massNumber of molelcules
Total (without water)987,86818
Polymers987,86818
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area47200 Å2
ΔGint-199 kcal/mol
Surface area200130 Å2
MethodPISA

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Components

#1: Protein
Gag polyprotein / Group-specific antigen


Mass: 54881.535 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Equine infectious anemia virus / Gene: gag / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P69730

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Equine infectious anemia virusEquine infectious anemia
Type: VIRUS
Details: Gag construct was expressed in E.coli and purified using the SUMO-tag system.
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Equine infectious anemia virus
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Details of virusEmpty: YES / Enveloped: NO / Isolate: OTHER / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Equus caballus
Virus shellName: Capsid / Diameter: 700 nm
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HClTris1
2100 mMSodium chlorideNaClSodium chloride1
32 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Virus-like-particles (tubular) of EIAV Gag deltaMA-deltap9 (referred to as Gag deltaMA) assembled at pH8.
Specimen supportDetails: 20 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 288 K / Details: 1-2 seconds blot time, offset -3mm

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Nanoprobe
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: -5000 nm / Nominal defocus min: -1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.8 sec. / Electron dose: 3.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1
Details: Data was acquired using a dose-symmetric tilt acquisition scheme, as described in Hagen et al, 2017, J. Struct. Biol, 197(2):191-8
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 3708 / Height: 3838 / Movie frames/image: 20

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Processing

EM software
IDNameVersionCategoryDetails
1MATLABvolume selectionpartially based on the TOM toolbox
2SerialEMimage acquisition
4CTFFIND4CTF correction
5CTFPHASEFLIPCTF correction
6NOVACTFCTF correction
9UCSF Chimeramodel fittingChimera was used to perform a rigid body fitting of one monomer of 2EIA into the electron microscopy density
10Cootmodel fittingThe last residues of CA and the first residues of SP (T347-L359) were manually built in Coot
13AV3final Euler assignment
14TOM Toolboxfinal Euler assignment
16AV33D reconstruction
17TOM Toolbox3D reconstruction
18PHENIXmodel refinement
19Cootmodel refinement
Image processingDetails: Tilt series were low-pass filtered according to their cumulative dose using exposure filters that were calculated using an exposure-dependent amplitude attenuation function and critical ...Details: Tilt series were low-pass filtered according to their cumulative dose using exposure filters that were calculated using an exposure-dependent amplitude attenuation function and critical exposure constants (as published in Grant & Grigorieff, Elife, 2015). Tilt series were aligned and reconstructed in IMOD.
CTF correctionDetails: CTF-correction was initially performed using ctfphaseflip in IMOD and NovaCTF in the final steps
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 89860 / Symmetry type: POINT
EM volume selectionMethod: Subvolumes were defined according to their position on the VLPs
Details: Subtomogram extraction positions were defined in Amira using the electron microscopy toolbox by determing the radii and the spline of the VLPs. Initially, positions were oversampled and ...Details: Subtomogram extraction positions were defined in Amira using the electron microscopy toolbox by determing the radii and the spline of the VLPs. Initially, positions were oversampled and subsequently cleaned during alignments using cross-correlation and distance thresholds.
Num. of tomograms: 20 / Num. of volumes extracted: 308367
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Rigid body fitting was done in Chimera. Missing residues were built de novo in Coot. Refinement was performed iteratively in Phenix and Coot.
Atomic model buildingPDB-ID: 2EIA

2eia

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