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- PDB-6hwy: Mature MLV capsid pentamer structure in intact virus particles -

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Basic information

Entry
Database: PDB / ID: 6hwy
TitleMature MLV capsid pentamer structure in intact virus particles
ComponentsPutative gag polyproteinGroup-specific antigen
KeywordsVIRAL PROTEIN / MLV / capsid / pentamer
Function / homologyCore shell protein Gag P30 / Zinc knuckle / Zinc finger CCHC-type profile. / Gag P30 core shell protein / Gag polyprotein, inner coat protein p12 / Gamma-retroviral matrix protein / Zinc finger, CCHC-type / Gag polyprotein, inner coat protein p12 / Matrix protein (MA), p15 / Retrovirus capsid, N-terminal ...Core shell protein Gag P30 / Zinc knuckle / Zinc finger CCHC-type profile. / Gag P30 core shell protein / Gag polyprotein, inner coat protein p12 / Gamma-retroviral matrix protein / Zinc finger, CCHC-type / Gag polyprotein, inner coat protein p12 / Matrix protein (MA), p15 / Retrovirus capsid, N-terminal / Retroviral matrix protein / Zinc finger, CCHC-type superfamily / Gamma-retroviral matrix domain superfamily / virion assembly / viral capsid / nucleic acid binding / host cell plasma membrane / structural molecule activity / zinc ion binding / membrane / Putative gag polyprotein
Function and homology information
Specimen sourceMurine leukemia virus
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / 8.6 Å resolution
AuthorsQu, K. / Glass, B. / Dolezal, M. / Schur, F.K.M. / Rein, A. / Rumlova, M. / Ruml, T. / Kraeusslich, H.G. / Briggs, J.A.G.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 15, 2018 / Release: Dec 5, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 5, 2018Structure modelrepositoryInitial release
1.1Dec 19, 2018Structure modelData collection / Database referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Assembly

Deposited unit
A: Putative gag polyprotein
B: Putative gag polyprotein
C: Putative gag polyprotein
D: Putative gag polyprotein


Theoretical massNumber of molelcules
Total (without water)108,6614
Polyers108,6614
Non-polymers00
Water0
1
A: Putative gag polyprotein
B: Putative gag polyprotein
C: Putative gag polyprotein
D: Putative gag polyprotein
x 5


Theoretical massNumber of molelcules
Total (without water)543,30620
Polyers543,30620
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation4

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Components

#1: Protein/peptide
Putative gag polyprotein / Group-specific antigen


Mass: 27165.305 Da / Num. of mol.: 4 / Source: (gene. exp.) Murine leukemia virus / Gene: gag / Cell line (production host): HEK 293T / Production host: Homo sapiens (human) / References: UniProt: A0A240FAQ8

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Murine leukemia virus / Type: VIRUS / Entity ID: 1 / Source: RECOMBINANT
Source (natural)Organism: Murine leukemia virus
Source (recombinant)Cell: HEK 293T / Organism: Homo sapiens (human) / Plasmid: M2204
Details of virusEmpty: NO / Enveloped: YES / Virus isolate: STRAIN / Virus type: VIRION
Buffer solutionpH: 6
SpecimenDetails: Purified virus solution was inactivated and diluted 1:1 with PBS containing 10 nm colloidal gold.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 288 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 / Nominal defocus max: 7500 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 microns / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.6 sec. / Electron dose: 1.8 e/Å2
Details: Dose fluctuation was caused by the ring collapse of FEG during data collection.
Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Number of grids imaged: 2
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 microns / Width: 3710 / Height: 3838 / Movie frames/image: 6 / Used frames/image: 1-6

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Processing

EM software
IDNameCategoryDetails
1AmiraFPMvolume selectionVolume selection
2MATLABvolume selectionVolume extraction
3SerialEMimage acquisitionDose-symmetric tilt-scheme
5MATLABCTF correctionCTF determination
6IMODCTF correctionPhase flipping only
9Cootmodel fitting
10UCSF Chimeramodel fitting
13AV3final Euler assignment
14TOMfinal Euler assignment
16AV33D reconstruction
17TOM3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C5
3D reconstructionResolution: 8.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 1299 / Algorithm: BACK PROJECTION / Symmetry type: POINT
EM volume selectionNumber of tomograms: 65 / Number of volumes extracted: 1299
Atomic model buildingRef protocol: RIGID BODY FIT / Ref space: REAL
Atomic model building
IDPDB-IDPdb chain ID 3D fitting IDPdb chain residue range
11U7KA11-131
26GZAA11-87

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