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- EMDB-22452: Adeno-Associated Virus Helicase domain Heptamer with ssDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-22452
TitleAdeno-Associated Virus Helicase domain Heptamer with ssDNA
Map dataLocal refined map of Helicase domain heptamer
Sample
  • Complex: Helicase domain heptamer of Rep68 complx bound to ssDNA
    • Protein or peptide: Protein Rep68
    • DNA: DNA (5'-D(P*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 / 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: 6.7 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: 0.29
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.29
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jsf
  • Surface level: 0.29
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22452.png

Downloads & links

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Map

FileDownload / File: emd_22452.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal refined map of Helicase domain heptamer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 400 pix.
= 330. Å		0.83 Å/pix.
x 400 pix.
= 330. Å		0.83 Å/pix.
x 400 pix.
= 330. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.29 / Movie #1: 0.29
Minimum - Maximum-0.10432456 - 0.7186311
Average (Standard dev.)0.009062048 (±0.03118189)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 330.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8250.8250.825
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z330.000330.000330.000
α/β/γ90.00090.00090.000
start NX/NY/NZ1081340
NX/NY/NZ13584349
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.1040.7190.009

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Supplemental data

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Sample components

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Entire : Helicase domain heptamer of Rep68 complx bound to ssDNA

EntireName: Helicase domain heptamer of Rep68 complx bound to ssDNA
Components
  • Complex: Helicase domain heptamer of Rep68 complx bound to ssDNA
    • Protein or peptide: Protein Rep68
    • DNA: DNA (5'-D(P*TP*TP*TP*T)-3')

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Supramolecule #1: Helicase domain heptamer of Rep68 complx bound to ssDNA

SupramoleculeName: Helicase domain heptamer of Rep68 complx bound to ssDNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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: 7 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Adeno-associated virus - 2
Molecular weightTheoretical: 60.908703 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: GPPGFYEIVI KVPSDLDEHL PGISDSFVNW VAEKEWELPP DSDMDLNLIE QAPLTVAEKL QRDFLTEWRR VSKAPEALFF VQFEKGESY FHMHVLVETT GVKSMVLGRF LSQIREKLIQ RIYRGIEPTL PNWFAVTKTR NGAGGGNKVV DESYIPNYLL P KTQPELQW ...String:
GPPGFYEIVI 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(P*TP*TP*TP*T)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*T)-3') / type: dna / ID: 2 / Number of copies: 1 / 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)aminomethane
200.0 mMNaClsodium chloride
1.0 mMC9H15O6PTris(2-carboxyethyl)phosphine
0.05 %C14H28O6Octyl-glucopyranosine
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AMYLAMINE
VitrificationCryogen name: ETHANE

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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 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 reconstructionResolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 46031
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: OTHER

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Atomic model buiding 1

Initial modelPDB ID:

1s9h


Chain - Residue range: 225-490 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-7jsf:
Adeno-Associated Virus Helicase domain Heptamer with ssDNA

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