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- EMDB-12496: Structure of the mature RSV CA lattice: Group II, hexamer-hexamer... -

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Basic information

Entry
Database: EMDB / ID: EMD-12496
TitleStructure of the mature RSV CA lattice: Group II, hexamer-hexamer interface, class 2'6
Map dataRSV CA lattice: hexamer-hexamer, class 2'6'
Sample
  • Virus: Rous sarcoma virus - Prague C
    • Protein or peptide: Capsid protein p27, alternate cleaved 1
KeywordsRetrovirus / Rous sarcoma virus / capsid protein / IP6 / VIRAL PROTEIN
Function / homology
Function and homology information


host cell nucleoplasm / viral procapsid maturation / host cell nucleolus / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / viral capsid / structural constituent of virion / nucleic acid binding / aspartic-type endopeptidase activity / viral translational frameshifting / host cell plasma membrane ...host cell nucleoplasm / viral procapsid maturation / host cell nucleolus / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / viral capsid / structural constituent of virion / nucleic acid binding / aspartic-type endopeptidase activity / viral translational frameshifting / host cell plasma membrane / proteolysis / zinc ion binding / membrane
Similarity search - Function
Retroviral Gag polyprotein, M / Retroviral M domain / : / gag protein p24 N-terminal domain / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic ...Retroviral Gag polyprotein, M / Retroviral M domain / : / gag protein p24 N-terminal domain / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Retroviral matrix protein / Retrovirus capsid, C-terminal / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily
Similarity search - Domain/homology
Biological speciesRous sarcoma virus (strain Prague C) / Rous sarcoma virus - Prague C
Methodsubtomogram averaging / cryo EM / Resolution: 6.7 Å
AuthorsObr M / Ricana CL
Funding support Austria, United States, European Union, 5 items
OrganizationGrant numberCountry
Austrian Science FundP31445 Austria
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI147890 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI150454 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R35GM136258 United States
European Union (EU)Horizon 2020, iNEXT (PID4246) , Grant number 653706European Union
CitationJournal: Nat Commun / Year: 2021
Title: Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer.
Authors: Martin Obr / Clifton L Ricana / Nadia Nikulin / Jon-Philip R Feathers / Marco Klanschnig / Andreas Thader / Marc C Johnson / Volker M Vogt / Florian K M Schur / Robert A Dick /
Abstract: Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold ...Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.
History
DepositionFeb 25, 2021-
Header (metadata) releaseApr 21, 2021-
Map releaseApr 21, 2021-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 7.2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 7.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nob
  • Surface level: 7.2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7nob
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12496.png

Downloads & links

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Map

FileDownload / File: emd_12496.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRSV CA lattice: hexamer-hexamer, class 2'6'
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.33 Å/pix.
x 192 pix.
= 254.938 Å		1.33 Å/pix.
x 192 pix.
= 254.938 Å		1.33 Å/pix.
x 192 pix.
= 254.938 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3278 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 7.2 / Movie #1: 7.2
Minimum - Maximum-17.003686999999999 - 32.286540000000002
Average (Standard dev.)0.01034607 (±2.4460166)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 254.9376 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.32780208333331.32780208333331.3278020833333
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z254.938254.938254.938
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-17.00432.2870.010

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Supplemental data

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Sample components

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Entire : Rous sarcoma virus - Prague C

EntireName: Rous sarcoma virus - Prague C
Components
  • Virus: Rous sarcoma virus - Prague C
    • Protein or peptide: Capsid protein p27, alternate cleaved 1

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Supramolecule #1: Rous sarcoma virus - Prague C

SupramoleculeName: Rous sarcoma virus - Prague C / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 11888 / Sci species name: Rous sarcoma virus - Prague C / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: unidentified (others)
Virus shellShell ID: 1 / Name: CANC polyhedra

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Macromolecule #1: Capsid protein p27, alternate cleaved 1

MacromoleculeName: Capsid protein p27, alternate cleaved 1 / type: protein_or_peptide / ID: 1 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Rous sarcoma virus (strain Prague C) / Strain: Prague C
Molecular weightTheoretical: 24.773594 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: PVVIKTEGPA WTPLEPKLIT RLADTVRTKG LRSPITMAEV EALMSSPLLP HDVTNLMRVI LGPAPYALWM DAWGVQLQTV IAAATRDPR HPANGQGRGE RTNLNRLKGL ADGMVGNPQG QAALLRPGEL VAITASALQA FREVARLAEP AGPWADIMQG P SESFVDFA ...String:
PVVIKTEGPA WTPLEPKLIT RLADTVRTKG LRSPITMAEV EALMSSPLLP HDVTNLMRVI LGPAPYALWM DAWGVQLQTV IAAATRDPR HPANGQGRGE RTNLNRLKGL ADGMVGNPQG QAALLRPGEL VAITASALQA FREVARLAEP AGPWADIMQG P SESFVDFA NRLIKAVEGS DLPPSARAPV IIDCFRQKSQ PDIQQLIRTA PSTLTTPGEI IKYVLDRQKT A

UniProtKB: Gag polyprotein

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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: 6.2
Component:
ConcentrationFormulaName
20.0 mMMES2-(N-morpholino)ethanesulfonic acid
100.0 mMNaClsodium chloride
2.0 nMTCEPtris(2-carboxyethyl)phosphine
100.0 microMIP6inositol hexakisphosphate
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 2.5 seconds blotting time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
DetailsAreas of interest for high-resolution data collection were identified in low magnification montages. Prior to tomogram acquisition, gain references were acquired and the filter was fully tuned. Microscope tuning was performed using the FEI AutoCTF software. The ilumination mode used during acquisition was nanoprobe.
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3708 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-10 / Number grids imaged: 1 / Average exposure time: 1.4 sec. / Average electron dose: 3.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
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

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Dynamo (ver. 1.1.333) / Number subtomograms used: 15152
ExtractionNumber tomograms: 49 / Number images used: 220000
Software: (Name: MATLAB (ver. R2018b), IMOD (ver. 4.9), Dynamo (ver. 1.1.333))
Details: The starting positions were defined by template matching. See materials and methods for details.
Final 3D classificationSoftware - Name: MATLAB (ver. R2018b)
Details: Subtomograms were sorted into classes based on their context in the lattice. See materials and methods for details
Final angle assignmentType: OTHER / Software - Name: Dynamo (ver. 1.1.333)
Details: Subtomogram alignment using Dynamo alignment project.

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Atomic model buiding 1

Initial modelPDB ID:

7no0


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-7nob:
Structure of the mature RSV CA lattice: Group II, hexamer-hexamer interface, class 2'6

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