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- EMDB-22167: Cryo-EM map of KSHV ORF68 -

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Basic information

Entry
Database: EMDB / ID: EMD-22167
TitleCryo-EM map of KSHV ORF68
Map dataORF68
Sample
  • Complex: Pentamer structure of KSHV ORF68
Function / homologyHerpesvirus major envelope glycoprotein / Herpesvirus putative major envelope glycoprotein / host cell cytoplasm / viral envelope / host cell nucleus / cytoplasm / Packaging protein UL32
Function and homology information
Biological speciesHuman gammaherpesvirus 8
Methodsingle particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsDidychuk AL / Gates SN / Martin A / Glaunsinger B
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI122528 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Elife / Year: 2021
Title: A pentameric protein ring with novel architecture is required for herpesviral packaging.
Authors: Allison L Didychuk / Stephanie N Gates / Matthew R Gardner / Lisa M Strong / Andreas Martin / Britt A Glaunsinger /
Abstract: Genome packaging in large double-stranded DNA viruses requires a powerful molecular motor to force the viral genome into nascent capsids, which involves essential accessory factors that are poorly ...Genome packaging in large double-stranded DNA viruses requires a powerful molecular motor to force the viral genome into nascent capsids, which involves essential accessory factors that are poorly understood. Here, we present structures of two such accessory factors from the oncogenic herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV; ORF68) and Epstein-Barr virus (EBV; BFLF1). These homologous proteins form highly similar homopentameric rings with a positively charged central channel that binds double-stranded DNA. Mutation of individual positively charged residues within but not outside the channel ablates DNA binding, and in the context of KSHV infection, these mutants fail to package the viral genome or produce progeny virions. Thus, we propose a model in which ORF68 facilitates the transfer of newly replicated viral genomes to the packaging motor.
History
DepositionJun 15, 2020-
Header (metadata) releaseFeb 24, 2021-
Map releaseFeb 24, 2021-
UpdateFeb 24, 2021-
Current statusFeb 24, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22167.png

Downloads & links

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Map

FileDownload / File: emd_22167.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationORF68
Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.06632936 - 0.15669547
Average (Standard dev.)0.0003705352 (±0.004242617)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 296.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.161.161.16
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z296.960296.960296.960
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ410410410
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0660.1570.000

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Supplemental data

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Sample components

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Entire : Pentamer structure of KSHV ORF68

EntireName: Pentamer structure of KSHV ORF68
Components
  • Complex: Pentamer structure of KSHV ORF68

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Supramolecule #1: Pentamer structure of KSHV ORF68

SupramoleculeName: Pentamer structure of KSHV ORF68 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Human gammaherpesvirus 8
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 536 KDa

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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

Concentration1.25 mg/mL
BufferpH: 7.6
Component:
ConcentrationNameFormula
50.0 mMHepes
50.0 mMSodium ChlorideNaClSodium chloride
50.0 mMPotassium ChlorideKCl
1.0 mMTCEP
1.0 %Glycerol
GridModel: C-flat / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20.0 nm
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: 2 second blot, 3 second wait.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 2408 / Average exposure time: 8.0 sec. / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 662435
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: Negative stain initial model
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 60772
FSC plot (resolution estimation)

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