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- EMDB-54429: Hexameric AAV2 Rep40-dsDNA (ITR) duplex complex in presence of ATPyS -

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Basic information

Entry
Database: EMDB / ID: EMD-54429
TitleHexameric AAV2 Rep40-dsDNA (ITR) duplex complex in presence of ATPyS
Map dataDeepEMhancer sharpened volume series map from continuous heterogeneity analysis (frame 18 component 0)
Sample
  • Complex: AAV2 Hexameric Rep40-dsDNA (ITR) duplex complex in presence of ATPyS
    • DNA: DNA
    • Protein or peptide: Protein Rep40
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsHelicase / Adeno-associated Virus / AAA+ ATPase / DNA BINDING PROTEIN
Function / homology
Function and homology information


viral genome replication / DNA helicase activity / DNA helicase / DNA replication / host cell nucleus / ATP hydrolysis activity / ATP binding
Similarity search - Function
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
Methodsingle particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsRouse SL / Bubeck D / Barritt JD / Xu V / Wake M
Funding support2 items
OrganizationGrant numberCountry
Other government
Other private
CitationJournal: Cell Rep / Year: 2026
Title: Structural basis for Rep-mediated adeno-associated virus packaging.
Authors: Victoria Xu / Aiysha McInnes / Matthew Wake / Marta Acebrón-García-de-Eulate / Joseph D Barritt / Doryen Bubeck / Sarah L Rouse /
Abstract: Adeno-associated viruses (AAVs) are parvoviruses utilized as gene therapy vectors. However, the AAV packaging mechanism is unresolved at the molecular level, creating a bottleneck for vector ...Adeno-associated viruses (AAVs) are parvoviruses utilized as gene therapy vectors. However, the AAV packaging mechanism is unresolved at the molecular level, creating a bottleneck for vector manufacturing, safety, and efficacy. Here, cryo-EM structures of the Rep helicase packaging motor in complex with the packaging marker DNA (ITR) and the Rep-AAV8 capsid complex are presented. Rep-ITR complexes reveal dynamic oligomeric states on the DNA, elucidating the strand separation mechanism coupled to its ATPase cycle. We observe Rep preferentially bound to empty capsids, with a binding interface likely conserved across the virus family. This complex also unveils a cryptic capsid ATP-binding site which, alongside Rep binding, triggers structural rearrangements priming the capsid for packaging. Collectively, these findings advance the understanding of Rep-mediated packaging, with significant implications for parvovirus virology and viral vector design.
History
DepositionJul 17, 2025-
Header (metadata) releaseFeb 4, 2026-
Map releaseFeb 4, 2026-
UpdateMar 18, 2026-
Current statusMar 18, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_54429.png

Downloads & links

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Map

FileDownload / File: emd_54429.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer sharpened volume series map from continuous heterogeneity analysis (frame 18 component 0)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 297. Å		0.83 Å/pix.
x 360 pix.
= 297. Å		0.83 Å/pix.
x 360 pix.
= 297. 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: 0.0536
Minimum - Maximum-0.0016976756 - 2.0686767
Average (Standard dev.)0.0012970576 (±0.02722867)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 297.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: non sharpened map

Fileemd_54429_additional_1.map
Annotationnon sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Consensus volume half map

Fileemd_54429_half_map_1.map
AnnotationConsensus volume half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Consensus volume half map

Fileemd_54429_half_map_2.map
AnnotationConsensus volume half 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 : AAV2 Hexameric Rep40-dsDNA (ITR) duplex complex in presence of ATPyS

EntireName: AAV2 Hexameric Rep40-dsDNA (ITR) duplex complex in presence of ATPyS
Components
  • Complex: AAV2 Hexameric Rep40-dsDNA (ITR) duplex complex in presence of ATPyS
    • DNA: DNA
    • Protein or peptide: Protein Rep40
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: AAV2 Hexameric Rep40-dsDNA (ITR) duplex complex in presence of ATPyS

SupramoleculeName: AAV2 Hexameric Rep40-dsDNA (ITR) duplex complex in presence of ATPyS
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Adeno-associated Virus 2 Replication protein 40 in complex with inverted-terminal-repeat dsDNA and ATP-gamma-S
Source (natural)Organism: adeno-associated virus 2

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Macromolecule #1: DNA

MacromoleculeName: DNA / type: dna / ID: 1
Details: Truncated dsDNA region of the full length AAV2 5'ITR
Number of copies: 1 / Classification: DNA
Source (natural)Organism: adeno-associated virus 2
Molecular weightTheoretical: 44.697359 KDa
SequenceString: (DT)(DT)(DG)(DG)(DC)(DC)(DA)(DC)(DT)(DC) (DC)(DC)(DT)(DC)(DT)(DC)(DT)(DG)(DC)(DG) (DC)(DG)(DC)(DT)(DC)(DG)(DC)(DT)(DC) (DG)(DC)(DT)(DC)(DA)(DC)(DT)(DG)(DA)(DG) (DG) (DC)(DC)(DG)(DG)(DG)(DC) ...String:
(DT)(DT)(DG)(DG)(DC)(DC)(DA)(DC)(DT)(DC) (DC)(DC)(DT)(DC)(DT)(DC)(DT)(DG)(DC)(DG) (DC)(DG)(DC)(DT)(DC)(DG)(DC)(DT)(DC) (DG)(DC)(DT)(DC)(DA)(DC)(DT)(DG)(DA)(DG) (DG) (DC)(DC)(DG)(DG)(DG)(DC)(DG)(DA) (DC)(DC)(DA)(DA)(DA)(DG)(DG)(DT)(DC)(DG) (DC)(DC) (DC)(DG)(DA)(DC)(DG)(DC)(DC) (DC)(DG)(DG)(DG)(DC)(DT)(DT)(DT)(DG)(DC) (DC)(DC)(DG) (DG)(DG)(DC)(DG)(DG)(DC) (DC)(DT)(DC)(DA)(DG)(DT)(DG)(DA)(DG)(DC) (DG)(DA)(DG)(DC) (DG)(DA)(DG)(DC)(DG) (DC)(DG)(DC)(DA)(DG)(DA)(DG)(DA)(DG)(DG) (DG)(DA)(DG)(DT)(DG) (DG)(DC)(DC)(DA) (DA)(DC)(DT)(DC)(DC)(DA)(DT)(DC)(DA)(DC) (DT)(DA)(DG)(DG)(DG)(DG) (DT)(DT)(DC) (DC)(DT)

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Macromolecule #2: Protein Rep40

MacromoleculeName: Protein Rep40 / type: protein_or_peptide / ID: 2 / Details: Latch-WTAAV2Rep40-TrB-3xFLAG-TEV-6xHis / Number of copies: 6 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: adeno-associated virus 2
Molecular weightTheoretical: 44.148922 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EQNKENQNPN SDAPVIRSKT SARYMELVGW LVDKGITSEK QWIQEDQASY ISFNAASNSR SQIKAALDNA GKIMSLTKTA PDYLVGQQP VEDISSNRIY KILELNGYDP QYAASVFLGW ATKKFGKRNT IWLFGPATTG KTNIAEAIAH TVPFYGCVNW T NENFPFND ...String:
EQNKENQNPN SDAPVIRSKT SARYMELVGW LVDKGITSEK QWIQEDQASY ISFNAASNSR SQIKAALDNA GKIMSLTKTA PDYLVGQQP VEDISSNRIY KILELNGYDP QYAASVFLGW ATKKFGKRNT IWLFGPATTG KTNIAEAIAH TVPFYGCVNW T NENFPFND CVDKMVIWWE EGKMTAKVVE SAKAILGGSK VRVDQKCKSS AQIDPTPVIV TSNTNMCAVI DGNSTTFEHQ QP LQDRMFK FELTRRLDHD FGKVTKQEVK DFFRWAKDHV VEVEHEFYVK KGGAKKRPAP SDADISEPKR VRESVAQPST SDA EASINY ADRLARGHSL GSLVPRGSGG GGSGGGGSGG GGSMDYKDHD GDYKDHDIDY KDDDDKGSEN LYFQSHHHHH H

UniProtKB: Protein Rep40

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Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 2 / 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 #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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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

Concentration0.8 mg/mL
BufferpH: 7.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 39.99 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Details: consensus refinement / Number images used: 108620
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-9s10:
Hexameric AAV2 Rep40-dsDNA (ITR) duplex complex in presence of ATPyS

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