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- EMDB-22451: Adeno-Associated Virus Origin Binding Domain in complex with ssDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-22451
TitleAdeno-Associated Virus Origin Binding Domain in complex with ssDNA
Map dataAAV-2 OBD-dT25 double octamer
Sample
  • Complex: AAV-2 Rep68-ssDNA complex
    • Complex: Poly thymine 25-mer
      • DNA: DNA (5'-D(*TP*TP*TP*T)-3')
    • Complex: AAV-2 Rep68
      • Protein or peptide: Protein Rep68
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 / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 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-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.23
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.23
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jse
  • Surface level: 0.28
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22451.png

Downloads & links

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Map

FileDownload / File: emd_22451.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAAV-2 OBD-dT25 double octamer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 400 pix.
= 329.92 Å		0.82 Å/pix.
x 400 pix.
= 329.92 Å		0.82 Å/pix.
x 400 pix.
= 329.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8248 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.23 / Movie #1: 0.23
Minimum - Maximum-0.2280611 - 0.87165755
Average (Standard dev.)0.013453689 (±0.046000306)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 329.92004 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.82480.82480.8248
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z329.920329.920329.920
α/β/γ90.00090.00090.000
start NX/NY/NZ937643
NX/NY/NZ114126230
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.2280.8720.013

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Supplemental data

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Sample components

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Entire : AAV-2 Rep68-ssDNA complex

EntireName: AAV-2 Rep68-ssDNA complex
Components
  • Complex: AAV-2 Rep68-ssDNA complex
    • Complex: Poly thymine 25-mer
      • DNA: DNA (5'-D(*TP*TP*TP*T)-3')
    • Complex: AAV-2 Rep68
      • Protein or peptide: Protein Rep68

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Supramolecule #1: AAV-2 Rep68-ssDNA complex

SupramoleculeName: AAV-2 Rep68-ssDNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: AAV-2 Rep68 in complex with poly-dT25
Source (natural)Organism: Adeno-associated virus - 2
Molecular weightTheoretical: 60.833 kDa/nm

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Supramolecule #2: Poly thymine 25-mer

SupramoleculeName: Poly thymine 25-mer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2 / Details: DNA
Source (natural)Organism: Adeno-associated virus - 2

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Supramolecule #3: AAV-2 Rep68

SupramoleculeName: AAV-2 Rep68 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1 / Details: 16 monomers in the complex
Source (natural)Organism: Adeno-associated virus - 2

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Macromolecule #1: Protein Rep68

MacromoleculeName: Protein Rep68 / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Adeno-associated virus - 2
Molecular weightTheoretical: 60.942789 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: GMPGFYEIVI KVPSDLDEHL PGISDSFVNW VAEKEWELPP DSDMDLNLIE QAPLTVAEKL QRDFLTEWRR VSKAPEALFF VQFEKGESY FHMHVLVETT GVKSMVLGRF LSQIREKLIQ RIYRGIEPTL PNWFAVTKTR NGAGGGNKVV DESYIPNYLL P KTQPELQW ...String:
GMPGFYEIVI KVPSDLDEHL PGISDSFVNW VAEKEWELPP DSDMDLNLIE QAPLTVAEKL QRDFLTEWRR VSKAPEALFF VQFEKGESY FHMHVLVETT GVKSMVLGRF LSQIREKLIQ RIYRGIEPTL PNWFAVTKTR NGAGGGNKVV DESYIPNYLL P KTQPELQW AWTNMEQYLS ACLNLTERKR LVAQHLTHVS QTQEQNKENQ NPNSDAPVIR SKTSARYMEL VGWLVDKGIT SE KQWIQED QASYISFNAA SNSRSQIKAA LDNAGKIMSL TKTAPDYLVG QQPVEDISSN RIYKILELNG YDPQYAASVF LGW ATKKFG KRNTIWLFGP ATTGKTNIAE AIAHTVPFYG CVNWTNENFP FNDCVDKMVI WWEEGKMTAK VVESAKAILG GSKV RVDQK CKSSAQIDPT PVIVTSNTNM CAVIDGNSTT FEHQQPLQDR MFKFELTRRL DHDFGKVTKQ EVKDFFRWAK DHVVE VEHE FYVKKGGAKK RPAPSDADIS EPKRVRESVA QPSTSDAEAS INYADRLARG HSL

UniProtKB: Protein Rep68

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

MacromoleculeName: DNA (5'-D(*TP*TP*TP*T)-3') / type: dna / ID: 2 / Number of copies: 8 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.171814 KDa
SequenceString:
(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

Concentration0.75 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
GridModel: C-flat / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Details: Emitech K950 unit with amylmine.
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: SUPER-RESOLUTION / Number real images: 1449 / Average exposure time: 8.0 sec. / Average electron dose: 92.03 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 165000 / 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: 118531
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 46031
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:

5byg


Chain - Chain ID: A / Chain - Residue range: 1-208 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient
Output model

PDB-7jse:
Adeno-Associated Virus Origin Binding Domain in complex with ssDNA

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