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- EMDB-22454: Adeno-Associated Virus 2 Rep68 HD Heptamer-ssAAVS1 with ATPgS -

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Basic information

Entry
Database: EMDB / ID: EMD-22454
TitleAdeno-Associated Virus 2 Rep68 HD Heptamer-ssAAVS1 with ATPgS
Map dataLocal refined map of heptameric Rep68 HD complex
Sample
  • Complex: Helicase domain heptamer of AAV-2 Rep68 complex bound to AAVS1 ssDNA.
    • Complex: AAV-2 Rep68
      • Protein or peptide: Protein Rep68
    • Complex: AAVS1 ssDNA
      • DNA: DNA (5'-D(P*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*C)-3')
KeywordsAAV / 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 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: 4.4 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 9, 2020-
Map releaseDec 9, 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: 0.413
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.413
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jsh
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22454.png

Downloads & links

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Map

FileDownload / File: emd_22454.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal refined map of heptameric Rep68 HD complex
Voxel sizeX=Y=Z: 1.41484 Å
Density
Contour LevelBy AUTHOR: 0.413 / Movie #1: 0.413
Minimum - Maximum-2.1726303 - 2.689523
Average (Standard dev.)-0.0020944097 (±0.089667395)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 362.19904 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.414839843751.414839843751.41483984375
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z362.199362.199362.199
α/β/γ90.00090.00090.000
start NX/NY/NZ937643
NX/NY/NZ114126230
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.1732.690-0.002

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Supplemental data

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Sample components

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Entire : Helicase domain heptamer of AAV-2 Rep68 complex bound to AAVS1 ssDNA.

EntireName: Helicase domain heptamer of AAV-2 Rep68 complex bound to AAVS1 ssDNA.
Components
  • Complex: Helicase domain heptamer of AAV-2 Rep68 complex bound to AAVS1 ssDNA.
    • Complex: AAV-2 Rep68
      • Protein or peptide: Protein Rep68
    • Complex: AAVS1 ssDNA
      • DNA: DNA (5'-D(P*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*C)-3')

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Supramolecule #1: Helicase domain heptamer of AAV-2 Rep68 complex bound to AAVS1 ssDNA.

SupramoleculeName: Helicase domain heptamer of AAV-2 Rep68 complex bound to AAVS1 ssDNA.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Model from local resolution electron density map of helicase domain.
Source (natural)Organism: Adeno-associated virus - 2
Molecular weightTheoretical: 4.481 kDa/nm

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Supramolecule #2: AAV-2 Rep68

SupramoleculeName: AAV-2 Rep68 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: Local map includes portion of the helicase domain
Source (natural)Organism: Adeno-associated virus - 2

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Supramolecule #3: AAVS1 ssDNA

SupramoleculeName: AAVS1 ssDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Protein Rep68

MacromoleculeName: Protein Rep68 / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Adeno-associated virus - 2
Molecular weightTheoretical: 61.00582 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: GPPPGFYEIV IKVPSDLDEH LPGISDSFVN WVAEKEWELP PDSDMDLNLI EQAPLTVAEK LQRDFLTEWR RVSKAPEALF FVQFEKGES YFHMHVLVET TGVKSMVLGR FLSQIREKLI QRIYRGIEPT LPNWFAVTKT RNGAGGGNKV VDESYIPNYL L PKTQPELQ ...String:
GPPPGFYEIV IKVPSDLDEH LPGISDSFVN WVAEKEWELP PDSDMDLNLI EQAPLTVAEK LQRDFLTEWR RVSKAPEALF FVQFEKGES YFHMHVLVET TGVKSMVLGR FLSQIREKLI QRIYRGIEPT LPNWFAVTKT RNGAGGGNKV VDESYIPNYL L PKTQPELQ WAWTNMEQYL SACLNLTERK RLVAQHLTHV SQTQEQNKEN QNPNSDAPVI RSKTSARYME LVGWLVDKGI TS EKQWIQE DQASYISFNA ASNSRSQIKA ALDNAGKIMS LTKTAPDYLV GQQPVEDISS NRIYKILELN GYDPQYAASV FLG WATKKF GKRNTIWLFG PATTGKTNIA EAIAHTVPFY GCVNWTNENF PFNDCVDKMV IWWEEGKMTA KVVESAKAIL GGSK VRVDQ KCKSSAQIDP TPVIVTSNTN MCAVIDGNST TFEHQQPLQD RMFKFELTRR LDHDFGKVTK QEVKDFFRWA KDHVV EVEH EFYVKKGGAK KRPAPSDADI SEPKRVRESV AQPSTSDAEA SINYADRLAR GHSL

UniProtKB: Protein Rep68

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Macromolecule #2: DNA (5'-D(P*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*C)-3')

MacromoleculeName: DNA (5'-D(P*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*CP*TP*CP*GP*C)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.497898 KDa
SequenceString:
(DC)(DG)(DC)(DT)(DC)(DG)(DC)(DT)(DC)(DG) (DC)(DT)(DC)(DG)(DC)

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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

Concentration0.75 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormulaName
25.0 mMC4H11NO32-Amino-2-(hydroxymethyl)propane-1,3-diol
200.0 mMNaClsodium chloride
1.0 mMC9H15O6PTris(2-carboxyethyl)phosphine hydrochloride
0.05 %C14H28O6Octyl-glucopyranoside
5.0 mMC10H12N5O12P3S1ATPgS
GridModel: C-flat / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number real images: 2669 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 59000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 433139
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 193869
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationSoftware - 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 / Target criteria: correlation coefficient
Output model

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

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