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- EMDB-44902: Cryo-EM Structure of AAV2 Rep68 bound to integration site AAVS1 -

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Basic information

Entry
Database: EMDB / ID: EMD-44902
TitleCryo-EM Structure of AAV2 Rep68 bound to integration site AAVS1
Map data
Sample
  • Complex: Rep68 AAVS1 ATPgS complex
    • Protein or peptide: Protein Rep68
    • DNA: DNA (21-MER)
    • DNA: DNA (21-MER)
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsAAV / Protein-DNA complex / Replication / Helicase / AAA+ / SF3 / DNA BINDING 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 / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity ...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 / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / 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 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.64 Å
AuthorsEscalante CR
Funding support United States, 1 items
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
DepositionMay 16, 2024-
Header (metadata) releaseFeb 12, 2025-
Map releaseFeb 12, 2025-
UpdateFeb 12, 2025-
Current statusFeb 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44902.png

Downloads & links

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Map

FileDownload / File: emd_44902.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.53 Å/pix.
x 600 pix.
= 316.8 Å		0.53 Å/pix.
x 600 pix.
= 316.8 Å		0.53 Å/pix.
x 600 pix.
= 316.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.528 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0116
Minimum - Maximum-0.113928206 - 0.14715979
Average (Standard dev.)0.00007564726 (±0.0023353163)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 316.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_44902_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44902_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Rep68 AAVS1 ATPgS complex

EntireName: Rep68 AAVS1 ATPgS complex
Components
  • Complex: Rep68 AAVS1 ATPgS complex
    • Protein or peptide: Protein Rep68
    • DNA: DNA (21-MER)
    • DNA: DNA (21-MER)
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Rep68 AAVS1 ATPgS complex

SupramoleculeName: Rep68 AAVS1 ATPgS complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Heptameric Rep68 complex bound to AAVS1 DNA site in the presence of ATPgS
Source (natural)Organism: adeno-associated virus 2
Molecular weightTheoretical: 458 KDa

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Macromolecule #1: Protein Rep68

MacromoleculeName: Protein Rep68 / type: protein_or_peptide / ID: 1 / Details: AAV-2 Rep68 (1-490) / Number of copies: 7 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: adeno-associated virus 2
Molecular weightTheoretical: 55.896184 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPPGFYEIVI KVPSDLDGHL PGISDSFVNW VAEKEWELPP DSDMDLNLIE QAPLTVAEKL QRDFLTEWRR VSKAPEALFF VQFEKGESY FHMHVLVETT GVKSMVLGRF LSQIREKLIQ RIYRGIEPTL PNWFAVTKTR NGAGGGNKVV DESYIPNYLL P KTQPELQW ...String:
GPPGFYEIVI KVPSDLDGHL 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 FYVKKGG

UniProtKB: Protein Rep68

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Macromolecule #2: DNA (21-MER)

MacromoleculeName: DNA (21-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.46313 KDa
SequenceString:
(DG)(DT)(DT)(DG)(DG)(DG)(DG)(DC)(DT)(DC) (DG)(DG)(DC)(DG)(DC)(DT)(DC)(DG)(DC)(DT) (DC)

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Macromolecule #3: DNA (21-MER)

MacromoleculeName: DNA (21-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.428152 KDa
SequenceString:
(DG)(DA)(DG)(DC)(DG)(DA)(DG)(DC)(DG)(DC) (DC)(DG)(DA)(DG)(DC)(DC)(DC)(DC)(DA)(DA) (DC)

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Macromolecule #4: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 3 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration4 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormulaName
25.0 mM(HOCH2)3CNH2tris(hydroxymethyl)aminomethane
200.0 mMNaClSodium Chloride
1.0 mMC9H15O6Ptris(2-carboxyethyl)phosphine
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AMYLAMINE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 8301 / 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 / Nominal defocus max: 2.103 µm / Nominal defocus min: 1.539 µm / Nominal magnification: 85000
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: 792736 / Details: Topaz picked particles
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4zq9


Details: OBD domain
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.3) / Number images used: 268936
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 2 / Avg.num./class: 312253
FSC plot (resolution estimation)

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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 / Details: Initial model was helicase domain of Rep40
SoftwareName: Coot (ver. 0.89)
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-Correlation coefficient
Output model

PDB-9bu7:
Cryo-EM Structure of AAV2 Rep68 bound to integration site AAVS1: Insights into the mechanism of DNA melting.

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