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- EMDB-4421: Representative tomogram of immature M-PMV particles -

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Basic information

Entry
Database: EMDB / ID: EMD-4421
TitleRepresentative tomogram of immature M-PMV particles
Map dataRepresentative tomogram of immature M-PMV particles for subtomogram averaging
Sample
  • Virus: Mason-Pfizer monkey virus
Biological speciesMason-Pfizer monkey virus
Methodelectron tomography / cryo EM
AuthorsQu K / Glass B / Dolezal M / Schur FKM / Rein A / Rumlova M / Ruml T / Kraeusslich HG / Briggs JAG
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Structure and architecture of immature and mature murine leukemia virus capsids.
Authors: Kun Qu / Bärbel Glass / Michal Doležal / Florian K M Schur / Brice Murciano / Alan Rein / Michaela Rumlová / Tomáš Ruml / Hans-Georg Kräusslich / John A G Briggs /
Abstract: Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as ...Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein-protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.
History
Header (metadata) releaseOct 24, 2018-
DepositionNov 14, 2018-
Map releaseDec 5, 2018-
UpdateDec 19, 2018-
Current statusDec 19, 2018Processing 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_4421.png

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Map

FileDownload / File: emd_4421.map.gz / Format: CCP4 / Size: 5 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationRepresentative tomogram of immature M-PMV particles for subtomogram averaging
Voxel sizeX=Y=Z: 2.7 Å
Density
Minimum - Maximum-1004. - 1004.
Average (Standard dev.)4.9462495 (±158.223890000000011)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-1-1-1
Dimensions18551855780
Spacing18551855780
CellA: 5008.5 Å / B: 5008.5 Å / C: 2106.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z2.72.72.7
M x/y/z18551855780
origin x/y/z0.0000.0000.000
length x/y/z5008.5005008.5002106.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-1-1-1
NC/NR/NS18551855780
D min/max/mean-1004.0001004.0004.946

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Supplemental data

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Sample components

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Entire : Mason-Pfizer monkey virus

EntireName: Mason-Pfizer monkey virus
Components
  • Virus: Mason-Pfizer monkey virus

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Supramolecule #1: Mason-Pfizer monkey virus

SupramoleculeName: Mason-Pfizer monkey virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1 / NCBI-ID: 11855 / Sci species name: Mason-Pfizer monkey virus / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host systemOrganism: Homo sapiens (human) / Recombinant cell: HEK 293T / Recombinant plasmid: pSHRM15 D26N

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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
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK II
DetailsPurified virus solution was inactivated and diluted 1:1 with PBS containing 10 nm colloidal gold.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: UMC Utrecht / Diameter: 10 nm

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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: 4.5 µm / Nominal defocus min: 2.0 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
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-6 / Number grids imaged: 1 / Average exposure time: 0.6 sec. / Average electron dose: 2.0 e/Å2
Details: Dose fluctuation was caused by the ring collapse of FEG during data collection.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware:
Namedetails
MATLABCTF determination
IMODPhase flipping only
Final reconstructionSoftware - Name: eTomo / Number images used: 41

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