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- EMDB-44424: AAV-2 Rep68-AAVS1 heptameric complex -

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Basic information

Entry
Database: EMDB / ID: EMD-44424
TitleAAV-2 Rep68-AAVS1 heptameric complex
Map data
Sample
  • Complex: AAV-2 Rep68-AAVS1 DNA complex
    • Protein or peptide: Protein Rep68
    • DNA: AAVS1 DNA (41-MER) Sense strand
    • DNA: AAVS1 DNA (41-MER) ANTISENSE
KeywordsAdeno-associated virus / non-structural protein / AAVS1 integration site / Protein-DNA complex / 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 speciesAAV-2 (virus) / adeno-associated virus 2 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.32 Å
AuthorsJaiswal R / Escalante 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
DepositionApr 7, 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_44424.png

Downloads & links

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Map

FileDownload / File: emd_44424.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 300 pix.
= 317.46 Å		1.06 Å/pix.
x 300 pix.
= 317.46 Å		1.06 Å/pix.
x 300 pix.
= 317.46 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0582 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.029
Minimum - Maximum-0.02083558 - 0.12535945
Average (Standard dev.)0.00024292387 (±0.005184531)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 317.46 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_44424_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_44424_half_map_2.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : AAV-2 Rep68-AAVS1 DNA complex

EntireName: AAV-2 Rep68-AAVS1 DNA complex
Components
  • Complex: AAV-2 Rep68-AAVS1 DNA complex
    • Protein or peptide: Protein Rep68
    • DNA: AAVS1 DNA (41-MER) Sense strand
    • DNA: AAVS1 DNA (41-MER) ANTISENSE

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Supramolecule #1: AAV-2 Rep68-AAVS1 DNA complex

SupramoleculeName: AAV-2 Rep68-AAVS1 DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Rep68 Heptameric complex on 50 bp AAVS1 DNA site.
Source (natural)Organism: AAV-2 (virus)
Molecular weightTheoretical: 458 KDa

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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: 55.968246 KDa
Recombinant expressionOrganism: synthetic construct (others)
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 FYVKKGG

UniProtKB: Protein Rep68

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Macromolecule #2: AAVS1 DNA (41-MER) Sense strand

MacromoleculeName: AAVS1 DNA (41-MER) Sense strand / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.62988 KDa
SequenceString:
(DG)(DG)(DC)(DG)(DG)(DG)(DT)(DG)(DG)(DT) (DG)(DG)(DC)(DG)(DG)(DC)(DG)(DG)(DT)(DT) (DG)(DG)(DG)(DG)(DC)(DT)(DC)(DG)(DG) (DC)(DG)(DC)(DT)(DC)(DG)(DC)(DT)(DC)(DG) (DC) (DT)(DC)(DG)(DC)(DT)(DG)(DG)(DG) (DC)(DG)

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Macromolecule #3: AAVS1 DNA (41-MER) ANTISENSE

MacromoleculeName: AAVS1 DNA (41-MER) ANTISENSE / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.190695 KDa
SequenceString:
(DC)(DG)(DC)(DC)(DC)(DA)(DG)(DC)(DG)(DA) (DG)(DC)(DG)(DA)(DG)(DC)(DG)(DA)(DG)(DC) (DG)(DC)(DC)(DG)(DA)(DG)(DC)(DC)(DC) (DC)(DA)(DA)(DC)(DC)(DG)(DC)(DC)(DG)(DC) (DC) (DA)(DC)(DC)(DA)(DC)(DC)(DC)(DG) (DC)(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

Concentration2 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormulaName
25.0 mM2-Amino-2-(hydroxymethyl)propane-1,3-diolTris.HCl
200.0 mMNaClSodium Chloride
1.0 mMtris(2-carboxyethyl)phosphineTCEP
GridModel: EMS Lacey Carbon / 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 / Details: GP2.
DetailsComplex purified by Size-exclusion Chromatography

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 8793 / Average exposure time: 2.5 sec. / Average electron dose: 26.23 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 81000
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: 3466434
Startup model#0 - Type of model: PDB ENTRY
#0 - PDB model - PDB ID:

4zq9


#1 - Type of model: PDB ENTRY
#1 - PDB model - PDB ID:

1s9h

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.32 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 450971
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

4zq9

residue_range: 1-208, source_name: PDB, initial_model_type: experimental modelOBD

1s9h

residue_range: 209-490, source_name: PDB, initial_model_type: experimental modelHD
RefinementProtocol: RIGID BODY FIT
Output model

PDB-9bc5:
AAV-2 Rep68-AAVS1 heptameric complex

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