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- EMDB-10383: A structure of the EIAV CA-SP hexamer (C2) from Gag-deltaMA tubes... -

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Basic information

Entry
Database: EMDB / ID: EMD-10383
TitleA structure of the EIAV CA-SP hexamer (C2) from Gag-deltaMA tubes assembled at pH6
Map dataA 3.8 Angstrom structure of the EIAV CA-SP hexamer (C2) from Gag-dMA tubes assembled at pH6
Sample
  • Virus: Equine infectious anemia virus
    • Protein or peptide: Gag polyproteinGroup-specific antigen
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
Methodsubtomogram averaging / cryo EM / Resolution: 3.8 Å
AuthorsDick RA / Xu C / Morado DR / Kravchuk V / Ricana CL / Lyddon TD / Broad AM / Feathers JR / Johnson MC / Vogt VM ...Dick RA / Xu C / Morado DR / Kravchuk V / Ricana CL / Lyddon TD / Broad AM / Feathers JR / Johnson MC / Vogt VM / Perilla JR / Briggs JAG / Schur FKM
Funding support Austria, United States, United Kingdom, Germany, 10 items
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, 2019-
Header (metadata) releaseJan 8, 2020-
Map releaseJan 15, 2020-
UpdateMar 30, 2022-
Current statusMar 30, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.599
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.599
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6t63
  • Surface level: 0.599
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6t63
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10383.png

Downloads & links

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Map

FileDownload / File: emd_10383.map.gz / Format: CCP4 / Size: 50.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationA 3.8 Angstrom structure of the EIAV CA-SP hexamer (C2) from Gag-dMA tubes assembled at pH6
Voxel sizeX=Y=Z: 1.041 Å
Density
Contour LevelBy AUTHOR: 0.499 / Movie #1: 0.599
Minimum - Maximum-1.5783836 - 2.1831098
Average (Standard dev.)0.00023123673 (±0.19501516)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions236236236
Spacing236236236
CellA=B=C: 245.676 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0411.0411.041
M x/y/z236236236
origin x/y/z0.0000.0000.000
length x/y/z245.676245.676245.676
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS236236236
D min/max/mean-1.5782.1830.000

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Supplemental data

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Sample components

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Entire : Equine infectious anemia virus

EntireName: Equine infectious anemia virus
Components
  • Virus: Equine infectious anemia virus
    • Protein or peptide: Gag polyproteinGroup-specific antigen

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Supramolecule #1: Equine infectious anemia virus

SupramoleculeName: Equine infectious anemia virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Gag construct was expressed in E.coli and purified using the SUMO-tag system. Assembly was performed at pH6.
NCBI-ID: 11665 / Sci species name: Equine infectious anemia virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Equus caballus (horse)
Host systemOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21
Virus shellShell ID: 1 / Name: Capsid / Diameter: 350.0 Å

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Macromolecule #1: Gag polyprotein

MacromoleculeName: Gag polyprotein / type: protein_or_peptide / ID: 1 / Number of copies: 18 / Enantiomer: LEVO
Source (natural)Organism: Equine infectious anemia virus
Molecular weightTheoretical: 54.881535 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGDPLTWSKA LKKLEKVTVQ GSQKLTTGNC NWALSLVDLF HDTNFVKEKD WQLRDVIPLL EDVTQTLSGQ EREAFERTWW AISAVKMGL QINNVVDGKA SFQLLRAKYE KKTANKKQSE PSEEYPIMID GAGNRNFRPL TPRGYTTWVN TIQTNGLLNE A SQNLFGIL ...String:
MGDPLTWSKA LKKLEKVTVQ GSQKLTTGNC NWALSLVDLF HDTNFVKEKD WQLRDVIPLL EDVTQTLSGQ EREAFERTWW AISAVKMGL QINNVVDGKA SFQLLRAKYE KKTANKKQSE PSEEYPIMID GAGNRNFRPL TPRGYTTWVN TIQTNGLLNE A SQNLFGIL SVDCTSEEMN AFLDVVPGQA GQKQILLDAI DKIADDWDNR HPLPNAPLVA PPQGPIPMTA RFIRGLGVPR ER QMEPAFD QFRQTYRQWI IEAMSEGIKV MIGKPKAQNI RQGAKEPYPE FVDRLLSQIK SEGHPQEISK FLTDTLTIQN ANE ECRNAM RHLRPEDTLE EKMYACRDIG TTKQKMMLLA KALQTGLAGP FKGGALKGGP LKAAQTCYNC GKPGHLSSQC RAPK VCFKC KQPGHFSKQC RSVPKNGKQG AQGRPQKQTF PIQQKSQHNK SVVQETPQTQ NLYPDLSEIK KEYNVKEKDQ VEDLN LDSL WE

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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
Component:
ConcentrationFormulaName
50.0 mMMES2-(N-morpholino)ethanesulfonic acid
100.0 mMNaClSodium chlorideSodium chloride
2.0 mMTCEPtris(2-carboxyethyl)phosphine
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 20mA
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 15 K / Instrument: FEI VITROBOT MARK II / Details: 1-2 seconds blot time, offset -3mm.
DetailsVirus-like-particles (tubular) of EIAV Gag deltaMAdeltap9 (referred to as Gag deltaMA) assembled at pH6.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -3.5 µm / Nominal defocus min: -1.5 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Detailsnanoprobe
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3708 pixel / Digitization - Dimensions - Height: 3838 pixel / Number grids imaged: 1 / Average exposure time: 1.4 sec. / Average electron dose: 3.4 e/Å2
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 56 / Number images used: 357324
Method: Subvolumes were defined according to their position on the VLPs
Software - Name: MATLAB / Software - details: partially based on the TOM toolbox
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.
CTF correctionSoftware: (Name: CTFFIND (ver. 4), NOVACTF) / Details: CTF-correction was performed using NOVACTF
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 1
Projection matching processing - Merit function: CC / Software: (Name: AV3, TOM Toolbox)
Details: Subtomogram alignment was performed as described in the published manuscript.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: AV3, TOM Toolbox) / Number subtomograms used: 112929
DetailsTilt 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.

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Atomic model buiding 1

Initial modelPDB ID:

2eia

DetailsRigid body fitting was done in Chimera. Missing residues were built de novo in Coot. Refinement was performed iteratively in Phenix and Coot.
RefinementSpace: REAL / Protocol: OTHER / Target criteria: Cross-correlation coefficient
Output model

PDB-6t63:
A model of the EIAV CA-SP hexamer (C2) from Gag-deltaMA tubes assembled at pH6

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