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- PDB-9bc5: AAV-2 Rep68-AAVS1 heptameric complex -

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Basic information

Entry
Database: PDB / ID: 9bc5
TitleAAV-2 Rep68-AAVS1 heptameric complex
Components
  • AAVS1 DNA (41-MER) ANTISENSE
  • AAVS1 DNA (41-MER) Sense strand
  • Protein Rep68
KeywordsVIRAL PROTEIN/DNA / Adeno-associated virus / non-structural protein / AAVS1 integration site / Protein-DNA complex / VIRAL PROTEIN / VIRAL PROTEIN-DNA complex
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
DNA / DNA (> 10) / Protein Rep68
Similarity search - Component
Biological speciesadeno-associated virus 2
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.32 Å
AuthorsJaiswal, R. / Escalante, C.R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)2R01GM124204 United States
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Cryo-EM structure of AAV2 Rep68 bound to integration site AAVS1: insights into the mechanism of DNA melting.
Authors: Rahul Jaiswal / Brandon Braud / Karen C Hernandez-Ramirez / Vishaka Santosh / Alexander Washington / Carlos R Escalante /
Abstract: The Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions necessary for the viral life cycle. During AAV DNA replication, Rep68 ...The Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions necessary for the viral life cycle. During AAV DNA replication, Rep68 assembles at the origin of replication, catalyzing the DNA melting and nicking reactions during the hairpin rolling replication process to complete the second-strand synthesis of the AAV genome. We report the cryo-electron microscopy structures of Rep68 bound to the adeno-associated virus integration site 1 in different nucleotide-bound states. In the nucleotide-free state, Rep68 forms a heptameric complex around DNA, with three origin-binding domains (OBDs) bound to the Rep-binding element sequence, while three remaining OBDs form transient dimers with them. The AAA+ domains form an open ring without interactions between subunits and DNA. We hypothesize that the heptameric structure is crucial for loading Rep68 onto double-stranded DNA. The ATPγS complex shows that only three subunits associate with the nucleotide, leading to a conformational change that promotes the formation of both intersubunit and DNA interactions. Moreover, three phenylalanine residues in the AAA+ domain induce a steric distortion in the DNA. Our study provides insights into how an SF3 helicase assembles on DNA and provides insights into the DNA melting process.
History
DepositionApr 7, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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: AAVS1 DNA (41-MER) Sense strand
I: AAVS1 DNA (41-MER) ANTISENSE


Theoretical massNumber of molelcules
Total (without water)422,5989
Polymers422,5989
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Protein Rep68


Mass: 55968.246 Da / Num. of mol.: 7 / Mutation: C151S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) adeno-associated virus 2 / Gene: Rep68 / Plasmid: PET-15b / Production host: synthetic construct (others) / References: UniProt: P03132, DNA helicase
#2: DNA chain AAVS1 DNA (41-MER) Sense strand


Mass: 15629.880 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: DNA chain AAVS1 DNA (41-MER) ANTISENSE


Mass: 15190.695 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
Has protein modificationN

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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: AAV-2 Rep68-AAVS1 DNA complex / Type: COMPLEX / Details: Rep68 Heptameric complex on 50 bp AAVS1 DNA site. / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.458 MDa / Experimental value: NO
Source (natural)Organism: AAV-2 (virus)
Source (recombinant)Organism: E.Coli (others) / Strain: BL21 pLys S / Plasmid: pet-15b
Buffer solutionpH: 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris.HCl2-Amino-2-(hydroxymethyl)propane-1,3-diol1
2200 mMSodium ChlorideNaCl1
31 mMTCEPtris(2-carboxyethyl)phosphine1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Complex purified by Size-exclusion Chromatography
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Details: GP2

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2900 nm / Nominal defocus min: 600 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 26.23 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 8793
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansMovie frames/image: 50

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Processing

EM softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3466434
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 450971 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeChain residue rangeDetailsInitial refinement model-IDPdb chain residue range
14zq9

4zq9

4zq91-208OBD11-208
21s9h

1s9h

1s9h209-490HD2209-490

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