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- PDB-7vfe: Cryo-EM structure of Vaccinia virus scaffolding protein D13 with ... -

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Basic information

Entry
Database: PDB / ID: 7vfe
TitleCryo-EM structure of Vaccinia virus scaffolding protein D13 with N-terminal polyhistidine tag
ComponentsScaffold protein D13
KeywordsVIRAL PROTEIN / Scaffold / capsid / double-jelly-roll
Function / homologyPoxvirus rifampicin-resistance / Poxvirus rifampicin resistance protein / response to antibiotic / identical protein binding / membrane / Scaffold protein OPG125
Function and homology information
Biological speciesVaccinia virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.63 Å
AuthorsWolf, M. / Hyun, J. / Matsunami, H. / Kim, T.G.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP18am0101076 Japan
Japan Society for the Promotion of Science (JSPS)JP17K07318 Japan
Japan Society for the Promotion of Science (JSPS)21K06039 Japan
CitationJournal: Nat Commun / Year: 2022
Title: Assembly mechanism of the pleomorphic immature poxvirus scaffold.
Authors: Jaekyung Hyun / Hideyuki Matsunami / Tae Gyun Kim / Matthias Wolf /
Abstract: In Vaccinia virus (VACV), the prototype poxvirus, scaffold protein D13 forms a honeycomb-like lattice on the viral membrane that results in formation of the pleomorphic immature virion (IV). The ...In Vaccinia virus (VACV), the prototype poxvirus, scaffold protein D13 forms a honeycomb-like lattice on the viral membrane that results in formation of the pleomorphic immature virion (IV). The structure of D13 is similar to those of major capsid proteins that readily form icosahedral capsids in nucleocytoplasmic large DNA viruses (NCLDVs). However, the detailed assembly mechanism of the nonicosahedral poxvirus scaffold has never been understood. Here we show the cryo-EM structures of the D13 trimer and scaffold intermediates produced in vitro. The structures reveal that the displacement of the short N-terminal α-helix is critical for initiation of D13 self-assembly. The continuous curvature of the IV is mediated by electrostatic interactions that induce torsion between trimers. The assembly mechanism explains the semiordered capsid-like arrangement of D13 that is distinct from icosahedral NCLDVs. Our structures explain how a single protein can self-assemble into different capsid morphologies and represent a local exception to the universal Caspar-Klug theory of quasi-equivalence.
History
DepositionSep 13, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 23, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 13, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jun 19, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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

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Structure visualization

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Assembly

Deposited unit
A: Scaffold protein D13
B: Scaffold protein D13
C: Scaffold protein D13


Theoretical massNumber of molelcules
Total (without water)192,4893
Polymers192,4893
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Scaffold protein D13 / 62 kDa protein / Rifampicin resistance protein


Mass: 64162.930 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus (strain Western Reserve)
Strain: Western Reserve / Gene: VACWR118, D13L / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P68440

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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: Vaccinia virus scaffolding protein D13 with N-terminal polyhistidine tag in its trimeric state
Type: COMPLEX
Details: Recombinant D13 was expressed with N-terminal polyhistidine-tag using bacterial expression system. The protein was purified using metal affinity chromatography and size exclusion ...Details: Recombinant D13 was expressed with N-terminal polyhistidine-tag using bacterial expression system. The protein was purified using metal affinity chromatography and size exclusion chromatography. The final purified protein was trimeric.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.195 MDa / Experimental value: NO
Source (natural)Organism: Vaccinia virus WR
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris hydrochlorideNH2C(CH2OH)3HCl1
2600 mMSodium chlrorideNaCl1
350 mML-arginineH2NC(NH)NH(CH2)3CH(NH2)CO2H1
450 mML-glutamic acidHO2CCH2CH2CH(NH2)CO2H1
52 mM2-mercapthoethanolHSCH2CH2OH1
SpecimenConc.: 0.12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 90 % / Chamber temperature: 277 K
Details: 3 microliter sample volume was loaded onto a holey grid with additional graphene oxide film. 10 sec waiting time, 5 sec blotting time and blot force 0, no delay time were applied before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 155000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 31 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1418
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.18rc7_3834: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION3.1particle selection
2EPUimage acquisition
4CTFFIND4.1.9CTF correctionCTF determination
5RELION3.1CTF correctionCTF correction and refinement
8UCSF Chimera1.13.1model fitting
9Coot0.9.1model fitting
11PHENIX1.18model refinement
12RELION3.1initial Euler assignment
13RELION3.1final Euler assignment
14RELION3.1classification
15RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 284340
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.63 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173354 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6BEI

6bei


Accession code: 6BEI / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00812981
ELECTRON MICROSCOPYf_angle_d0.56617664
ELECTRON MICROSCOPYf_dihedral_angle_d15.7334761
ELECTRON MICROSCOPYf_chiral_restr0.0522031
ELECTRON MICROSCOPYf_plane_restr0.0052262

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