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- EMDB-22455: Adeno-Associated Virus 2 Rep68 HD Hexamer-ssDNA with ATPgS -

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Basic information

Entry
Database: EMDB / ID: EMD-22455
TitleAdeno-Associated Virus 2 Rep68 HD Hexamer-ssDNA with ATPgS
Map dataLocal Refinement of helicase domain hexamer bound to ssDNA
Sample
  • Complex: AAV-2 Rep68/dT25 complex
    • Protein or peptide: Protein Rep68
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*T)-3')
KeywordsAAV / Protein-DNA / AAA+ / SF3 / HUH / VIRAL PROTEIN
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 replication / DNA helicase / 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 replication / DNA helicase / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.01 Å
AuthorsEscalante CR
Funding support United States, 1 items
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, 2020-
Header (metadata) releaseDec 16, 2020-
Map releaseDec 16, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.34
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.34
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jsi
  • Surface level: 0.34
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22455.png

Downloads & links

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Map

FileDownload / File: emd_22455.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal Refinement of helicase domain hexamer bound to ssDNA
Voxel sizeX=Y=Z: 1.073 Å
Density
Contour LevelBy AUTHOR: 0.34 / Movie #1: 0.34
Minimum - Maximum-0.4025626 - 1.1557715
Average (Standard dev.)0.0071188994 (±0.06389815)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 214.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0731.0731.073
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z214.600214.600214.600
α/β/γ90.00090.00090.000
start NX/NY/NZ1081340
NX/NY/NZ13584349
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.4031.1560.007

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Supplemental data

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Sample components

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Entire : AAV-2 Rep68/dT25 complex

EntireName: AAV-2 Rep68/dT25 complex
Components
  • Complex: AAV-2 Rep68/dT25 complex
    • Protein or peptide: Protein Rep68
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*T)-3')

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Supramolecule #1: AAV-2 Rep68/dT25 complex

SupramoleculeName: AAV-2 Rep68/dT25 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: hexameric complex
Source (natural)Organism: Adeno-associated virus - 2
Molecular weightTheoretical: 370 KDa

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Macromolecule #1: Protein Rep68

MacromoleculeName: Protein Rep68 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Adeno-associated virus - 2
Molecular weightTheoretical: 60.829742 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MPGFYEIVIK VPSDLDGHLP GISDSFVNWV AEKEWELPPD SDMDLNLIEQ APLTVAEKLQ RDFLTEWRRV SKAPEALFFV QFEKGESYF HMHVLVETTG VKSMVLGRFL SQIREKLIQR IYRGIEPTLP NWFAVTKTRN GAGGGNKVVD ECYIPNYLLP K TQPELQWA ...String:
MPGFYEIVIK VPSDLDGHLP GISDSFVNWV AEKEWELPPD SDMDLNLIEQ APLTVAEKLQ RDFLTEWRRV SKAPEALFFV QFEKGESYF HMHVLVETTG VKSMVLGRFL SQIREKLIQR IYRGIEPTLP NWFAVTKTRN GAGGGNKVVD ECYIPNYLLP K TQPELQWA WTNMEQYLSA CLNLTERKRL VAQHLTHVSQ TQEQNKENQN PNSDAPVIRS KTSARYMELV GWLVDKGITS EK QWIQEDQ ASYISFNAAS NSRSQIKAAL DNAGKIMSLT KTAPDYLVGQ QPVEDISSNR IYKILELNGY DPQYAASVFL GWA TKKFGK RNTIWLFGPA TTGKTNIAEA IAHTVPFYGC VNWTNENFPF NDCVDKMVIW WEEGKMTAKV VESAKAILGG SKVR VDQKC KSSAQIDPTP VIVTSNTNMC AVIDGNSTTF EHQQPLQDRM FKFELTRRLD HDFGKVTKQE VKDFFRWAKD HVVEV EHEF YVKKGGAKKR PAPSDADISE PKRVRESVAQ PSTSDAEASI NYADRLARGH SL

UniProtKB: Protein Rep68

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Macromolecule #2: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*T)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*T)-3') / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.388585 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)

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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

Concentration0.5 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormulaName
25.0 mMC4H11NO3(hydroxymethyl) aminomethane
200.0 mMNaClSodium Chloride
1.0 mMC9H15O6PTCEP
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AMYLAMINE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1722 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 578679
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.01 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 61195
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial modelPDB ID:

1s9h


Chain - Chain ID: a / Chain - Residue range: 225-490 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7jsi:
Adeno-Associated Virus 2 Rep68 HD Hexamer-ssDNA with ATPgS

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