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- EMDB-31950: Cryo-EM structure of Vaccinia virus scaffolding protein D13 with ... -

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Basic information

Entry
Database: EMDB / ID: EMD-31950
TitleCryo-EM structure of Vaccinia virus scaffolding protein D13 with N-terminal polyhistidine tag
Map dataCryo-EM single particle reconstruction on Vaccinia virus scaffold protein D13 trimer with N-terminal poly-histidine tag. The map has been sharpened and density-normalized.
Sample
  • Complex: Vaccinia virus scaffolding protein D13 with N-terminal polyhistidine tag in its trimeric state
    • Protein or peptide: Scaffold protein D13
KeywordsScaffold / capsid / double-jelly-roll / VIRAL PROTEIN
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 WR / Vaccinia virus (strain Western Reserve)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.63 Å
AuthorsWolf M / Hyun J
Funding support Japan, 3 items
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, 2021-
Header (metadata) releaseFeb 23, 2022-
Map releaseFeb 23, 2022-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7vfe
  • Surface level: 5
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7vfe
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31950.png

Downloads & links

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Map

FileDownload / File: emd_31950.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM single particle reconstruction on Vaccinia virus scaffold protein D13 trimer with N-terminal poly-histidine tag. The map has been sharpened and density-normalized.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 256 pix.
= 265.216 Å		1.04 Å/pix.
x 256 pix.
= 265.216 Å		1.04 Å/pix.
x 256 pix.
= 265.216 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.036 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.0 / Movie #1: 5
Minimum - Maximum-21.521726999999998 - 37.893154000000003
Average (Standard dev.)0.000000002432808 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 265.216 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0361.0361.036
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z265.216265.216265.216
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-21.52237.8930.000

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Supplemental data

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Mask #1

Fileemd_31950_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Additional map: Cryo-EM single particle reconstruction on Vaccinia virus scaffold...

Fileemd_31950_additional_1.map
AnnotationCryo-EM single particle reconstruction on Vaccinia virus scaffold protein D13 trimer with N-terminal poly-histidine tag. The map is unsharpened and density-normalized.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cryo-EM single particle reconstruction on Vaccinia virus scaffold...

Fileemd_31950_additional_2.map
AnnotationCryo-EM single particle reconstruction on Vaccinia virus scaffold protein D13 trimer with N-terminal poly-histidine tag. The map is density-modified and density-normalized.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM single particle reconstruction on Vaccinia virus scaffold...

Fileemd_31950_half_map_1.map
AnnotationCryo-EM single particle reconstruction on Vaccinia virus scaffold protein D13 trimer with N-terminal poly-histidine tag (half map 1).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM single particle reconstruction on Vaccinia virus scaffold...

Fileemd_31950_half_map_2.map
AnnotationCryo-EM single particle reconstruction on Vaccinia virus scaffold protein D13 trimer with N-terminal poly-histidine tag (half map 2).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Vaccinia virus scaffolding protein D13 with N-terminal polyhistid...

EntireName: Vaccinia virus scaffolding protein D13 with N-terminal polyhistidine tag in its trimeric state
Components
  • Complex: Vaccinia virus scaffolding protein D13 with N-terminal polyhistidine tag in its trimeric state
    • Protein or peptide: Scaffold protein D13

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Supramolecule #1: Vaccinia virus scaffolding protein D13 with N-terminal polyhistid...

SupramoleculeName: Vaccinia virus scaffolding protein D13 with N-terminal polyhistidine tag in its trimeric state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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.
Source (natural)Organism: Vaccinia virus WR
Molecular weightTheoretical: 195 KDa

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Macromolecule #1: Scaffold protein D13

MacromoleculeName: Scaffold protein D13 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Vaccinia virus (strain Western Reserve) / Strain: Western Reserve
Molecular weightTheoretical: 64.16293 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: HHHHHHDYDI PTTENLYFQG AMNNTIINSL IGGDDSIKRS NVFAVDSQIP TLYMPQYISL SGVMTNDGPD NQAIASFEIR DQYITALNH LVLSLELPEV KGMGRFGYVP YVGYKCINHV SISSCNGVIW EIEGEELYNN CINNTIALKH SGYSSELNDI S IGLTPNDT ...String:
HHHHHHDYDI PTTENLYFQG AMNNTIINSL IGGDDSIKRS NVFAVDSQIP TLYMPQYISL SGVMTNDGPD NQAIASFEIR DQYITALNH LVLSLELPEV KGMGRFGYVP YVGYKCINHV SISSCNGVIW EIEGEELYNN CINNTIALKH SGYSSELNDI S IGLTPNDT IKEPSTVYVY IKTPFDVEDT FSSLKLSDSK ITVTVTFNPV SDIVIRDSSF DFETFNKEFV YVPELSFIGY MV KNVQIKP SFIEKPRRVI GQINQPTATV TEVHAATSLS VYTKPYYGNT DNKFISYPGY SQDEKDYIDA YVSRLLDDLV IVS DGPPTG YPESAEIVEV PEDGIVSIQD ADVYVKIDNV PDNMSVYLHT NLLMFGTRKN SFIYNISKKF SAITGTYSDA TKRT IFAHI SHSINIIDTS IPVSLWTSQR NVYNGDNRSA ESKAKDLFIN DPFIKGIDFK NKTDIISRLE VRFGNDVLYS ENGPI SRIY NELLTKSNNG TRTLTFNFTP KIFFRPTTIT ANVSRGKDKL SVRVVYSTMD VNHPIYYVQK QLVVVCNDLY KVSYDQ GVS ITKIMG

UniProtKB: Scaffold protein OPG125

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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

Concentration0.12 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMNH2C(CH2OH)3HClTris hydrochloride
600.0 mMNaClSodium chlroride
50.0 mMH2NC(NH)NH(CH2)3CH(NH2)CO2HL-arginine
50.0 mMHO2CCH2CH2CH(NH2)CO2HL-glutamic acid
2.0 mMHSCH2CH2OH2-mercapthoethanol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1418 / Average exposure time: 31.0 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 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 155000
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: 284340
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.63 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 173354
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 72197 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6bei


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7vfe:
Cryo-EM structure of Vaccinia virus scaffolding protein D13 with N-terminal polyhistidine tag

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