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- EMDB-10386: Cryo-electron tomogram containing EIAV Gag-deltaMA spheres and tu... -

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Entry
Database: EMDB / ID: EMD-10386
TitleCryo-electron tomogram containing EIAV Gag-deltaMA spheres and tubes assembled at pH6
Map dataCryo-electron tomogram containing EIAV Gag-deltaMA spheres and tubes assembled at pH6
Sample
  • Virus: Equine infectious anemia virus
Biological speciesEquine infectious anemia virus
Methodelectron tomography / cryo EM
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-
UpdateNov 18, 2020-
Current statusNov 18, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_10386.png

Downloads & links

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Map

FileDownload / File: emd_10386.map.gz / Format: CCP4 / Size: 135.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-electron tomogram containing EIAV Gag-deltaMA spheres and tubes assembled at pH6
Voxel sizeX=Y=Z: 8.328 Å
Density
Minimum - Maximum-3434.792 - 3399.958
Average (Standard dev.)103.01864 (±83.679924)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0050
Dimensions464464165
Spacing464464165
CellA: 3864.1921 Å / B: 3864.1921 Å / C: 1374.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z8.3288.3288.328
M x/y/z464464165
origin x/y/z0.0000.0000.000
length x/y/z3864.1923864.1921374.120
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS0050
NC/NR/NS464464165
D min/max/mean-3434.7923399.958103.019

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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. 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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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 6
Component:
ConcentrationFormulaName
50.0 mMMES2-(N-morpholino)ethanesulfonic acid
100.0 mMNaClSodium 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.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: BBI / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -3.5 µm / Nominal defocus min: -1.5 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

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.
Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 41
CTF correctionSoftware: (Name: CTFFIND (ver. 4), NOVACTF) / Details: CTF-correction was performed using NOVACTF

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