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- PDB-7jsf: Adeno-Associated Virus Helicase domain Heptamer with ssDNA -

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Basic information

Entry
Database: PDB / ID: 7jsf
TitleAdeno-Associated Virus Helicase domain Heptamer with ssDNA
Components
  • DNA (5'-D(P*TP*TP*TP*T)-3')
  • Protein Rep68
KeywordsVIRAL PROTEIN / AAV / Protein-DNA / AAA+ / SF3 / HUH
Function / homology
Function and homology information


symbiont-mediated arrest of host cell cycle during G2/M transition / symbiont entry into host cell via permeabilization of host membrane / viral DNA genome replication / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / endonuclease activity / DNA helicase / DNA replication / host cell nucleus / ATP hydrolysis activity / DNA binding ...symbiont-mediated arrest of host cell cycle during G2/M transition / symbiont entry into host cell via permeabilization of host membrane / viral DNA genome replication / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / endonuclease activity / DNA helicase / DNA replication / host cell nucleus / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding
Similarity search - Function
Rep protein catalytic-like / Rep protein catalytic domain like / : / Parvovirus (PV) NS1 nuclease (NS1-Nuc) domain profile. / Parvovirus non-structural protein 1, helicase domain / Parvovirus non-structural protein NS1 / Helicase, superfamily 3, DNA virus / Superfamily 3 helicase of DNA viruses domain profile. / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesAdeno-associated virus - 2
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsEscalante, C.R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124204 United States
CitationJournal: Nucleic Acids Res / Year: 2020
Title: The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states.
Authors: Vishaka Santosh / Faik N Musayev / Rahul Jaiswal / Francisco Zárate-Pérez / Bram Vandewinkel / Caroline Dierckx / Molly Endicott / Kamyar Sharifi / Kelly Dryden / Els Henckaerts / Carlos R Escalante /
Abstract: The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and ...The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to 'catch' the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.
History
DepositionAug 14, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 16, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 23, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Protein Rep68
B: Protein Rep68
C: Protein Rep68
D: Protein Rep68
E: Protein Rep68
F: Protein Rep68
G: Protein Rep68
H: DNA (5'-D(P*TP*TP*TP*T)-3')


Theoretical massNumber of molelcules
Total (without water)427,5338
Polymers427,5338
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Protein Rep68


Mass: 60908.703 Da / Num. of mol.: 7 / Mutation: C151S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus - 2 / Gene: Rep68
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P03132, DNA helicase
#2: DNA chain DNA (5'-D(P*TP*TP*TP*T)-3')


Mass: 1171.814 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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: Helicase domain heptamer of Rep68 complx bound to ssDNA
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Adeno-associated virus - 210804
21synthetic construct (others)32630
Buffer solutionpH: 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM2-amino-2-(hydroxymethyl)aminomethaneC4H11NO31
2200 mMsodium chlorideNaCl1
31 mMTris(2-carboxyethyl)phosphineC9H15O6P1
40.05 %Octyl-glucopyranosineC14H28O61
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 165000 X
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 92.03 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1449

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Processing

EM software
IDNameCategory
2Leginonimage acquisition
7PHENIXmodel fitting
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 118531
3D reconstructionResolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 46031 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 1S9H

1s9h


Accession code: 1S9H / Pdb chain residue range: 225-490 / Source name: PDB / Type: experimental model

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