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

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Basic information

Entry
Database: PDB / ID: 7no0
TitleStructure of the mature RSV CA lattice: T=1 CA icosahedron
ComponentsCapsid protein p27, alternate cleaved 1
KeywordsVIRAL PROTEIN / Retrovirus / Rous sarcoma virus / capsid protein / IP6
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 / Retrovirus capsid C-terminal domain / Human Immunodeficiency Virus Type 1 Capsid Protein / Human Immunodeficiency Virus Type 1 Capsid Protein / Non-ribosomal Peptide Synthetase Peptidyl Carrier Protein; Chain A / : / gag protein p24 N-terminal domain / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal ...Retroviral Gag polyprotein, M / Retroviral M domain / Retrovirus capsid C-terminal domain / Human Immunodeficiency Virus Type 1 Capsid Protein / Human Immunodeficiency Virus Type 1 Capsid Protein / Non-ribosomal Peptide Synthetase Peptidyl Carrier Protein; Chain A / : / 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 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Biological speciesRous sarcoma virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsObr, M. / Ricana, C.L. / Nikulin, N. / Feathers, J.-P.R. / Klanschnig, M. / Thader, A. / Johnson, M.C. / Vogt, V.M. / Schur, F.K.M. / Dick, R.A.
Funding support Austria, United States, 4items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 21, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 9, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jul 10, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Assembly

Deposited unit
A: Capsid protein p27, alternate cleaved 1


Theoretical massNumber of molelcules
Total (without water)24,7741
Polymers24,7741
Non-polymers00
Water00
1
A: Capsid protein p27, alternate cleaved 1
x 60


Theoretical massNumber of molelcules
Total (without water)1,486,41660
Polymers1,486,41660
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
MethodUCSF CHIMERA

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Components

#1: Protein Capsid protein p27, alternate cleaved 1


Mass: 24773.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rous sarcoma virus (strain Prague C) / Strain: Prague C / Gene: gag / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03322

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Rous sarcoma virus - Prague C / Type: VIRUS / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Rous sarcoma virus - Prague C
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Details of virusEmpty: YES / Enveloped: NO / Isolate: OTHER / Type: VIRUS-LIKE PARTICLE
Virus shellName: T=1 icosahedron
Buffer solutionpH: 8
Buffer componentConc.: 1 M / Name: sodium phosphate / Formula: NaPi
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K / Details: blot time= 2.5 s blot force= 0

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 63000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
4GctfCTF correction
5RELIONCTF correction
8UCSF Chimeramodel fitting
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
14PHENIXmodel refinement
15Cootmodel refinement
CTF correctionDetails: CTF estimation and correction was performed using GCTF in the RELION wrapper
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles 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.
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 20498 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: The 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 ...Details: The 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.
Atomic model buildingPDB-ID: 3TIR

3tir


Accession code: 3TIR / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00515170
ELECTRON MICROSCOPYf_angle_d0.67720675
ELECTRON MICROSCOPYf_dihedral_angle_d16.4252085
ELECTRON MICROSCOPYf_chiral_restr0.0432390
ELECTRON MICROSCOPYf_plane_restr0.0042720

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