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- PDB-9ikc: Orf virus scaffolding protein Orfv075 -

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Basic information

Entry
Database: PDB / ID: 9ikc
TitleOrf virus scaffolding protein Orfv075
Components62 kDa protein
KeywordsVIRAL PROTEIN / Scaffold protein / Capsid protein / Poxvirus
Function / homologyPoxvirus rifampicin-resistance / Poxvirus rifampicin resistance protein / response to antibiotic / 62 kDa protein
Function and homology information
Biological speciesOrf virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.9 Å
AuthorsHyun, J. / Kim, S. / Ko, S. / Kim, M. / Jang, Y.
Funding support Korea, Republic Of, 1items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)2022R1A2C1005885 Korea, Republic Of
CitationJournal: Biochem Biophys Res Commun / Year: 2024
Title: Cryo-EM structure of orf virus scaffolding protein orfv075.
Authors: Seungmi Kim / Sumin Ko / Minjae Kim / Yeontae Jang / Jaekyung Hyun /
Abstract: Capsid-like poxvirus scaffold proteins self-assemble into semi-regular lattice that govern the formation of spherical immature virus particles. The scaffolding is a critical step in virus ...Capsid-like poxvirus scaffold proteins self-assemble into semi-regular lattice that govern the formation of spherical immature virus particles. The scaffolding is a critical step in virus morphogenesis as exemplified by the drug rifampicin that impairs the recruitment of scaffold onto the viral membrane in vaccinia virus (VACV). Here we report cryo-electron microscopy structure of scaffolding protein Orfv075 of orf virus (ORFV) that causes smallpox-like diseases in sheep, goats and occasionally humans via zoonotic infection. We demonstrate that the regions that are involved in intertrimeric interactions for scaffold assembly are largely conserved in comparison to its VACV orthologue protein D13 whose intermediate assembly structures have been previously characterized. By contrast, less conserved regions are located away from these interfaces, indicating both viruses share similar assembly mechanisms. We also show that the phenylalanine-rich binding site of rifampicin in D13 is conserved in Orfv075, and molecular docking simulation confirms similar binding modes. Our study provides structural basis of scaffolding protein as a target for anti-poxvirus treatment across wide range of poxvirus genera.
History
DepositionJun 27, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 7, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 62 kDa protein
B: 62 kDa protein
C: 62 kDa protein


Theoretical massNumber of molelcules
Total (without water)190,9573
Polymers190,9573
Non-polymers00
Water2,702150
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein 62 kDa protein / Rifampicin resistance protein


Mass: 63652.172 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Orf virus (strain NZ2) / Gene: 01orf_00068 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6TW25
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 150 / Source method: isolated from a natural source / Formula: H2O

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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: Orfv075 trimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.19 MDa / Experimental value: NO
Source (natural)Organism: Orf virus (strain NZ2)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3) / Plasmid: pPROEX-Hta
Details of virusIsolate: STRAIN / Type: VIRION
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HClC4H11NO31
2500 mMSodium chlorideNaCl1
350 mML-arginineC6H14N4O2C6H14N4O21
450 mML-glutamic acidC5H9NO41
52 mMBeta-mercapthoethanolC2H6OS1
SpecimenConc.: 0.13 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 / Humidity: 90 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 76923 X / Nominal defocus max: 1800 nm / Nominal defocus min: 700 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 / Temperature (max): 93 K / Temperature (min): 77 K
Image recordingAverage exposure time: 2.6 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10975
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.4.1particle selectionCryoSPARC blob picker was ued for initial particle picking followed by template-based picking from 2D class averages as a template.
2EPU3.6image acquisitionAutomated data collection was performed using EPU software with AFIS scheme.
4cryoSPARC4.4.1CTF correctionCryoSPARC Patch CTF was used for CTF estimation.
7Coot0.9.8.92model fitting
9cryoSPARC4.4.1initial Euler assignmentCryoSPARC ab-initio reconstruction and heterogenous refinement were used to assign initial 3D orientation.
10cryoSPARC4.4.1final Euler assignmentCryoSPARC homogeneous refinement was used for final 3D orientation assignment.
12cryoSPARC4.4.13D reconstructionCryoSPARC homogeneous refinement was used for final 3D reconstruction.
13PHENIX1.21model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2051025
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 1.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 714562 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Structure of orthologue protein D13 (PDB 7VFD) was fitted into the density map of orfv075 using UCSF Chimera. The fitted structure was modified in Coot based on sequence alignment between ...Details: Structure of orthologue protein D13 (PDB 7VFD) was fitted into the density map of orfv075 using UCSF Chimera. The fitted structure was modified in Coot based on sequence alignment between D13 and Orfv075. The resulting Orfv075 model was refined using PHENIX.
Atomic model buildingPDB-ID: 7VFD

7vfd


Pdb chain-ID: A / Accession code: 7VFD / Chain residue range: 1-547 / Pdb chain residue range: 1-547 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00212669
ELECTRON MICROSCOPYf_angle_d0.46317229
ELECTRON MICROSCOPYf_dihedral_angle_d4.4561733
ELECTRON MICROSCOPYf_chiral_restr0.0452001
ELECTRON MICROSCOPYf_plane_restr0.0042223

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