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- EMDB-10382: A 3.9 Angstrom structure of the EIAV CA-SP hexamer (C6) from Gag-... -

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Entry
Database: EMDB / ID: EMD-10382
TitleA 3.9 Angstrom structure of the EIAV CA-SP hexamer (C6) from Gag-dMA spheres assembled at pH8
Map dataA 3.9 Angstrom structure of the EIAV CA-SP hexamer (C6) from Gag-dMA spheres assembled at pH8
Sample
  • Virus: Equine infectious anemia virus
Biological speciesEquine infectious anemia virus
Methodsubtomogram averaging / cryo EM / Resolution: 3.9 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 Germany, Austria, United States, United Kingdom, 10 items
OrganizationGrant numberCountry
German Research FoundationBR 3635/2-1 Germany
Austrian Science FundP31445 Austria
National Institutes of Health/National Institute of General Medical SciencesR01-GM107013 United States
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesR01-AI147890 United States
National Institutes of Health/National Institute of General Medical SciencesP30-GM110758 United States
National Science Foundation (United States)1659534 United States
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesAI142263 United States
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesP50AI150481 United States
Medical Research Council (United Kingdom)MC_UP_1201/16 United Kingdom
European Research CouncilERC-2014-CoG 648432 - MEMBRANEFUSION United Kingdom
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-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.135
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.135
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10382.png

Downloads & links

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Map

FileDownload / File: emd_10382.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationA 3.9 Angstrom structure of the EIAV CA-SP hexamer (C6) from Gag-dMA spheres assembled at pH8
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.135 / Movie #1: 0.135
Minimum - Maximum-0.39861804 - 0.5326841
Average (Standard dev.)0.00028072987 (±0.044402953)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 259.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z259.200259.200259.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.3990.5330.000

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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

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Supramolecule #1: Equine infectious anemia virus

SupramoleculeName: Equine infectious anemia virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Gag construct was expressed in E.coli and purified using the SUMO-tag system
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: 1000.0 Å

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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: 8
Component:
ConcentrationFormulaName
50.0 mMTris-HClTris
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: 20 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK II
Details: 1-2 seconds blot time, offset -3mm 10 nm colloidal gold was added prior to vitrification.
DetailsVirus-like-particles (spherical) of EIAV Gag deltaMAdeltap9 (referred to as Gag deltaMA) assembled at pH8.

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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: -5.0 µm / Nominal defocus min: -1.5 µm / Nominal magnification: 105000
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: 1.8 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: 37 / Number images used: 191612 / 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 center 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 center of the VLPs. Initially, positions were oversampled and subsequently cleaned during alignments using cross-correlation and distance thresholds.
CTF correctionSoftware: (Name: CTFFIND (ver. 4), CTFPHASEFLIP, NOVACTF)
Details: CTF-correction was initially performed using ctfphaseflip in IMOD and NovaCTF in the final steps
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: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: AV3, TOM Toolbox) / Number subtomograms used: 65807
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

RefinementSpace: REAL

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