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- EMDB-31954: Cryo-EM structure of Vaccinia virus scaffolding protein D13 trime... -

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Basic information

Entry
Database: EMDB / ID: EMD-31954
TitleCryo-EM structure of Vaccinia virus scaffolding protein D13 trimer sextet
Map dataCryo-EM single particle reconstruction on Vaccinia virus scaffold protein D13 trimer sextet. The map has been sharpened using Relion post-processing, and density-normalized (mean=0, s.d=1).
Sample
  • Complex: Vaccinia virus scaffolding protein D13 with N-terminal 17 residue truncation, in its trimer sextet oligomeric 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 / membrane / identical protein binding / Scaffold protein OPG125
Function and homology information
Biological speciesVaccinia virus WR / Vaccinia virus (strain Western Reserve)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 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: 4.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7vfh
  • Surface level: 4.5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31954.png

Downloads & links

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Map

FileDownload / File: emd_31954.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 sextet. The map has been sharpened using Relion post-processing, and density-normalized (mean=0, s.d=1).
Voxel sizeX=Y=Z: 1.39 Å
Density
Contour LevelBy AUTHOR: 4.5 / Movie #1: 4.5
Minimum - Maximum-9.366586 - 23.475874000000001
Average (Standard dev.)-0.000000003216903 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 355.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.391.391.39
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z355.840355.840355.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-9.36723.476-0.000

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Supplemental data

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Mask #1

Fileemd_31954_msk_1.map
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_31954_additional_1.map
AnnotationCryo-EM single particle reconstruction on Vaccinia virus scaffold protein D13 trimer sextet. The map is unsharpened, and density-normalized (mean=0, s.d=1).
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_31954_additional_2.map
AnnotationCryo-EM single particle reconstruction on Vaccinia virus scaffold protein D13 trimer sextet. The map has been density-modified using PHENIX ResolveCryoEM, and density-normalized (mean=0, s.d=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_31954_half_map_1.map
AnnotationCryo-EM single particle reconstruction on Vaccinia virus scaffold protein D13 trimer sextet (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_31954_half_map_2.map
AnnotationCryo-EM single particle reconstruction on Vaccinia virus scaffold protein D13 trimer sextet (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 17 residue...

EntireName: Vaccinia virus scaffolding protein D13 with N-terminal 17 residue truncation, in its trimer sextet oligomeric state
Components
  • Complex: Vaccinia virus scaffolding protein D13 with N-terminal 17 residue truncation, in its trimer sextet oligomeric state
    • Protein or peptide: Scaffold protein D13

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Supramolecule #1: Vaccinia virus scaffolding protein D13 with N-terminal 17 residue...

SupramoleculeName: Vaccinia virus scaffolding protein D13 with N-terminal 17 residue truncation, in its trimer sextet oligomeric state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Recombinant D13 was expressed with N-terminal polyhistidine-tag (His-tag) using bacterial expression system. The protein was purified using metal affinity chromatography. His-tag was removed ...Details: Recombinant D13 was expressed with N-terminal polyhistidine-tag (His-tag) using bacterial expression system. The protein was purified using metal affinity chromatography. His-tag was removed by proteolysis and the protein was further purified using size exclusion chromatography. The final purified protein was trimeric. D13 assembled in tubular objects in low salt buffer.
Source (natural)Organism: Vaccinia virus WR
Molecular weightTheoretical: 1.080 MDa

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Macromolecule #1: Scaffold protein D13

MacromoleculeName: Scaffold protein D13 / type: protein_or_peptide / ID: 1 / Number of copies: 18 / Enantiomer: LEVO
Source (natural)Organism: Vaccinia virus (strain Western Reserve) / Strain: Western Reserve
Molecular weightTheoretical: 59.817219 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: RSNVFAVDSQ IPTLYMPQYI SLSGVMTNDG PDNQAIASFE IRDQYITALN HLVLSLELPE VKGMGRFGYV PYVGYKCINH VSISSCNGV IWEIEGEELY NNCINNTIAL KHSGYSSELN DISIGLTPND TIKEPSTVYV YIKTPFDVED TFSSLKLSDS K ITVTVTFN ...String:
RSNVFAVDSQ IPTLYMPQYI SLSGVMTNDG PDNQAIASFE IRDQYITALN HLVLSLELPE VKGMGRFGYV PYVGYKCINH VSISSCNGV IWEIEGEELY NNCINNTIAL KHSGYSSELN DISIGLTPND TIKEPSTVYV YIKTPFDVED TFSSLKLSDS K ITVTVTFN PVSDIVIRDS SFDFETFNKE FVYVPELSFI GYMVKNVQIK PSFIEKPRRV IGQINQPTAT VTEVHAATSL SV YTKPYYG NTDNKFISYP GYSQDEKDYI DAYVSRLLDD LVIVSDGPPT GYPESAEIVE VPEDGIVSIQ DADVYVKIDN VPD NMSVYL HTNLLMFGTR KNSFIYNISK KFSAITGTYS DATKRTIFAH ISHSINIIDT SIPVSLWTSQ RNVYNGDNRS AESK AKDLF INDPFIKGID FKNKTDIISR LEVRFGNDVL YSENGPISRI YNELLTKSNN GTRTLTFNFT PKIFFRPTTI TANVS RGKD KLSVRVVYST MDVNHPIYYV QKQLVVVCND LYKVSYDQGV SITKIMG

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
150.0 mMNaClSodium chlroride
2.0 mMHSCH2CH2OH2-mercapthoethanol
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
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: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 2 / Number real images: 7621 / Average exposure time: 10.0 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: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
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: 303052
Details: D13 trimer sextet images were extracted from the images of tubular assembly (EMD-31953) by symmetry expansion and signal subtraction.
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0) / Number images used: 247311
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM (ver. 1.0.0)
Final 3D classificationNumber classes: 3 / Avg.num./class: 101017 / Software - Name: cisTEM (ver. 1.0.0)
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-7vfh:
Cryo-EM structure of Vaccinia virus scaffolding protein D13 trimer sextet

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