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- EMDB-60655: Orf virus scaffolding protein Orfv075 -

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Basic information

Entry
Database: EMDB / ID: EMD-60655
TitleOrf virus scaffolding protein Orfv075
Map data3D reconstruction of Orfv075 (sharpened)
Sample
  • Complex: Orfv075 trimer
    • Protein or peptide: 62 kDa protein
  • Ligand: water
KeywordsScaffold protein / Capsid protein / Poxvirus / VIRAL PROTEIN
Function / homologyPoxvirus rifampicin-resistance / Poxvirus rifampicin resistance protein / response to antibiotic / 62 kDa protein
Function and homology information
Biological speciesOrf virus (strain NZ2)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.9 Å
AuthorsHyun J / Kim S / Ko S / Kim M / Jang Y
Funding support Korea, Republic Of, 1 items
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, 2024-
Header (metadata) releaseAug 7, 2024-
Map releaseAug 7, 2024-
UpdateAug 7, 2024-
Current statusAug 7, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60655.png

Downloads & links

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Map

FileDownload / File: emd_60655.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of Orfv075 (sharpened)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 384 pix.
= 249.6 Å		0.65 Å/pix.
x 384 pix.
= 249.6 Å		0.65 Å/pix.
x 384 pix.
= 249.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.09
Minimum - Maximum-0.3471265 - 0.6583064
Average (Standard dev.)0.000021669968 (±0.018351028)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 249.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_60655_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 3D reconstruction of Orfv075 (unsharpened)

Fileemd_60655_additional_1.map
Annotation3D reconstruction of Orfv075 (unsharpened)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: First halfmap of Orfv075 3D reconstruction

Fileemd_60655_half_map_1.map
AnnotationFirst halfmap of Orfv075 3D reconstruction
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Second halfmap of Orfv075 3D reconstruction

Fileemd_60655_half_map_2.map
AnnotationSecond halfmap of Orfv075 3D reconstruction
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Orfv075 trimer

EntireName: Orfv075 trimer
Components
  • Complex: Orfv075 trimer
    • Protein or peptide: 62 kDa protein
  • Ligand: water

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Supramolecule #1: Orfv075 trimer

SupramoleculeName: Orfv075 trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Orf virus (strain NZ2)
Molecular weightTheoretical: 190 KDa

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Macromolecule #1: 62 kDa protein

MacromoleculeName: 62 kDa protein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Orf virus (strain NZ2)
Molecular weightTheoretical: 63.652172 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSYYHHHHHH DYDIPTTENL YFQGAMNNSV VSLVGGDDAV RRASVFATDH RAPTVYMPQY ITTQGVVDTT SDAVTVTFEI RDKYISAMN NFVLSVDLPE IKGVGKMCYV PYIAYKLIRH VAVNSAADTI WETSGEELFD SCLDNERVME LSGFSRELND L STGSSPND ...String:
MSYYHHHHHH DYDIPTTENL YFQGAMNNSV VSLVGGDDAV RRASVFATDH RAPTVYMPQY ITTQGVVDTT SDAVTVTFEI RDKYISAMN NFVLSVDLPE IKGVGKMCYV PYIAYKLIRH VAVNSAADTI WETSGEELFD SCLDNERVME LSGFSRELND L STGSSPND VIKEAACVHA YIKTPFDADK TFSTLKLSDS KVTVTVTLNP VACVMVYDET FDAAKLAKEF PYSMELSFIG YM VKNLCPR PAFIEMPRRR VEQINHTTAV ITDVHACTSL SVYMKPVLSD ANNRFISYPG FQQSEGDFVM AFVERLLEDM VIV SNCYPE GFPETAEIVE VPPSGVVSIQ DTDVFVRIDD VPVGMRVFLH TNILVFATRK NSVVYNMSKK FSAITGAYSR ATSR IRFTT AIHSVNIGDA SVPVGVWTCQ RNVYNGDNRS PEARAKDLFV ADPFLKGVDF KNKIDVIARM DVRFGNEVLY SENSA VSRV FGEILGKTPG VRTLQFNFTP STFFSPTALN SNVSRGKDKL AVRVTTAHME AHNPLMYVPR QMVVVCNEVY RLSYDA GIV AEKVTAQ

UniProtKB: 62 kDa protein

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 150 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.13 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris-HCl
500.0 mMNaClSodium chloride
50.0 mMC6H14N4O2C6H14N4O2L-arginine
50.0 mMC5H9NO4L-glutamic acid
2.0 mMC2H6OSBeta-mercapthoethanol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 77.0 K / Max: 93.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 10975 / Average exposure time: 2.6 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 76923 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 130000
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

Particle selectionNumber selected: 2051025
Startup modelType of model: INSILICO MODEL
Details: Initial model was generated using CryoSPARC ab-initio model generation from the particles that had been assorted by 2D classification.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1)
Software - details: CryoSPARC homogeneous refinement was used for final 3D reconstruction.
Number images used: 714562
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Software - details: CryoSPARC ab-initio reconstruction and heterogenous refinement were used to assign initial 3D orientation.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Software - details: CryoSPARC homogeneous refinement was used for final 3D orientation assignment.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7vfd


Chain - Chain ID: A / Chain - Residue range: 1-547 / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsStructure 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.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-9ikc:
Orf virus scaffolding protein Orfv075

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