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- PDB-7jsh: Adeno-Associated Virus 2 Rep68 HD Heptamer-ssAAVS1 with ATPgS -

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Basic information

Entry
Database: PDB / ID: 7jsh
TitleAdeno-Associated Virus 2 Rep68 HD Heptamer-ssAAVS1 with ATPgS
Components
  • DNA (5'-D(P*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*C)-3')
  • Protein Rep68
KeywordsVIRAL PROTEIN/DNA / AAV / Protein-DNA / AAA+ / SF3 / HUH / VIRAL PROTEIN / VIRAL PROTEIN-DNA complex
Function / homology
Function and homology information


symbiont-mediated arrest of host cell cycle during G2/M transition / permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner 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 ...symbiont-mediated arrest of host cell cycle during G2/M transition / permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner 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
Parvovirus (PV) NS1 nuclease (NS1-Nuc) domain profile. / Rep protein catalytic-like / Rep protein catalytic domain like / 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
DNA / DNA (> 10) / Protein Rep68
Similarity search - Component
Biological speciesAdeno-associated virus - 2
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 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 9, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Dec 23, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 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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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-22454
  • Imaged by UCSF Chimera
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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
N: DNA (5'-D(P*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*C)-3')


Theoretical massNumber of molelcules
Total (without water)431,5398
Polymers431,5398
Non-polymers00
Water0
1


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

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Components

#1: Protein
Protein Rep68


Mass: 61005.820 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*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*C)-3')


Mass: 4497.898 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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Helicase domain heptamer of AAV-2 Rep68 complex bound to AAVS1 ssDNA.COMPLEXModel from local resolution electron density map of helicase domain.all0RECOMBINANT
2AAV-2 Rep68COMPLEXLocal map includes portion of the helicase domain#11RECOMBINANT
3AAVS1 ssDNACOMPLEX#21MULTIPLE SOURCES
Molecular weight
IDEntity assembly-IDValue (°)UnitsExperimental value
110.4245 MDaYES
210.425 MDaYES
31MEGADALTONSYES
414.481 kDa/nmYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Adeno-associated virus - 210804
21synthetic construct (others)32630
32Adeno-associated virus - 210804
43synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
22Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
Buffer solutionpH: 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM2-Amino-2-(hydroxymethyl)propane-1,3-diolC4H11NO31
2200 mMsodium chlorideNaClSodium chloride1
31 mMTris(2-carboxyethyl)phosphine hydrochlorideC9H15O6P1
40.05 %Octyl-glucopyranosideC14H28O61
55 mMATPgSC10H12N5O12P3S11
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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 FIELDBright-field microscopy / Calibrated magnification: 59000 X
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 2669

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
4CTFFIND4CTF correction
7PHENIXmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 433139
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 193869 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 1S9H

1s9h


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

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