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- EMDB-12485: Structure of the mature RSV CA lattice: T=1 CA icosahedron -

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Basic information

Entry
Database: EMDB / ID: EMD-12485
TitleStructure of the mature RSV CA lattice: T=1 CA icosahedron
Map dataRSV CA T=1 particle
Sample
  • Virus: Rous sarcoma virus - Prague C
    • Protein or peptide: Capsid protein p27, alternate cleaved 1
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 / host cell plasma membrane / proteolysis ...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 / 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 / Retrovirus capsid, C-terminal ...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 / Retrovirus capsid, C-terminal / Retroviral matrix protein / 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
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsObr M / Ricana CL / Nikulin N / Feathers J-PR / Klanschnig M / Thader A / Johnson MC / Vogt VM / Schur FKM / Dick RA
Funding support Austria, United States, 4 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
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-
UpdateJun 9, 2021-
Current statusJun 9, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7no0
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7no0
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12485.png

Downloads & links

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Map

FileDownload / File: emd_12485.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRSV CA T=1 particle
Voxel sizeX=Y=Z: 1.24 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.08270606 - 0.17907338
Average (Standard dev.)0.0002749659 (±0.003916739)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 555.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.241.241.24
M x/y/z448448448
origin x/y/z0.0000.0000.000
length x/y/z555.520555.520555.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS448448448
D min/max/mean-0.0830.1790.000

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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 systemOrganism: Escherichia coli BL21(DE3) (bacteria)
Virus shellShell ID: 1 / Name: T=1 icosahedron

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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: 1 / 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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8 / Component - Concentration: 1.0 M / Component - Formula: NaPi / Component - Name: sodium phosphate
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot time= 2.5 s blot force= 0.

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Electron beamAcceleration voltage: 200 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: 1.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 63000
Specialist opticsEnergy filter - Name: GIF Bioquantum / 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 K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average exposure time: 3.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 150599
Details: 2394 micrographs were taken, from which 374 particles were manually picked and 2D classified to generate templates for auto-picking. Two rounds of auto-picking and 2D classification resulted ...Details: 2394 micrographs were taken, from which 374 particles were manually picked and 2D classified to generate templates for auto-picking. Two rounds of auto-picking and 2D classification resulted in 150599 extracted particles.
CTF correctionSoftware: (Name: Gctf, RELION)
Details: CTF estimation and correction was performed using GCTF in the RELION wrapper
Startup modelType of model: EMDB MAP
EMDB ID:

5772


Details: Map was low pass filtered to 60 Angstrom^-1
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Details: 2D and 3D classification, CTF refinement and Bayesian particle polishing
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 20498

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Atomic model buiding 1

Initial modelPDB ID:

3tir

DetailsThe CANTD and CACTD of one CA monomer of pdb 3TIR were independently placed into the EM-density using the rigid body fitting option in UCSF Chimera. Subsequently the linker connecting the two CA domains was joined in Coot. To account for the different monomer-monomer interactions in the RSV CA pentamer, the monomers were replicated according to the inherent 5-fold symmetry of the map and an additional ring of CACTDs was rigid-body fitted into the EM-densities of the surrounding CA pentamers. A map segment (defined by a mask extending 3 Angstrom around the rigid body fitted model) was extracted, and real-space coordinate refinement against the EM-density was performed using Phenix. This was iterated with manual model building in Coot, similar as described previously. In brief, secondary structure restraints and non-crystallographic symmetry (NCS) restraints were applied throughout all refinements. Each Phenix iteration consisted of 5 macro cycles, in which simulated annealing was performed in every macro cycle. Atomic displacement parameter (ADP) refinement was per formed at the end of each iteration.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7no0:
Structure of the mature RSV CA lattice: T=1 CA icosahedron

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