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- PDB-6xfa: Cryo-EM structure of EBV BFLF1 -

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Basic information

Entry
Database: PDB / ID: 6xfa
TitleCryo-EM structure of EBV BFLF1
ComponentsPackaging protein UL32Packaging and labeling
KeywordsVIRAL PROTEIN / viral packaging / viral cleavage
Function / homologyHerpesvirus major envelope glycoprotein / Herpesvirus putative major envelope glycoprotein / host cell cytoplasm / viral envelope / host cell nucleus / cytoplasm / Packaging protein UL32 / Packaging protein UL32 homolog
Function and homology information
Biological speciesEpstein-Barr virus (Epstein-Barr virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsDidychuk, A.L. / Gates, S.N. / Martin, A. / Glaunsinger, B.
Funding support United States, 2items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 24, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Packaging protein UL32
B: Packaging protein UL32
C: Packaging protein UL32
D: Packaging protein UL32
E: Packaging protein UL32
F: Packaging protein UL32
G: Packaging protein UL32
H: Packaging protein UL32
I: Packaging protein UL32
J: Packaging protein UL32
hetero molecules


Theoretical massNumber of molelcules
Total (without water)538,09630
Polymers536,78810
Non-polymers1,30820
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25810 Å2
ΔGint-148 kcal/mol
Surface area157910 Å2

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Components

#1: Protein
Packaging protein UL32 / Packaging and labeling


Mass: 53678.797 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Epstein-Barr virus (strain GD1) (Epstein-Barr virus)
Strain: GD1 / Gene: BFLF1 / Production host: Homo sapiens (human) / References: UniProt: A0A2D1LYN0, UniProt: P03184*PLUS
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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: Decamer structure of EBV BFLF1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.536 MDa / Experimental value: NO
Source (natural)Organism: Epstein-Barr virus (strain GD1) (Epstein-Barr virus)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
160 mMHepes1
2100 mMSodium ChlorideNaClSodium chloride1
350 mMPotassium ChlorideKCl1
410 mMMagnesium ChlorideMgCl21
50.5 mMTCEP1
60.05 %NP-401
SpecimenConc.: 4 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: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 295 K / Details: 2 second blot, 3 second wait

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 2.4 sec. / Electron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 839

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 278234
SymmetryPoint symmetry: D5 (2x5 fold dihedral)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32272 / Symmetry type: POINT

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