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- EMDB-54403: Consensus map of Hexameric AAV2 Rep40-dsDNA (ITR) duplex complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-54403
TitleConsensus map of Hexameric AAV2 Rep40-dsDNA (ITR) duplex complex in presence of ATPyS
Map data
Sample
  • Complex: AAV2 Hexameric Rep40-dsDNA (ITR) duplex complex in presence of ATPyS
KeywordsHelicase / Adeno-associated Virus / AAA+ ATPase / DNA BINDING PROTEIN
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 15, 2025-
Header (metadata) releaseMar 18, 2026-
Map releaseMar 18, 2026-
UpdateMar 18, 2026-
Current statusMar 18, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_54403.png

Downloads & links

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Map

FileDownload / File: emd_54403.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 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: 0.0668
Minimum - Maximum-0.27015483 - 0.51489264
Average (Standard dev.)-0.00034422948 (±0.011975106)
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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Half map: #1

Fileemd_54403_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_54403_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 : 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

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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
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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

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) / Number images used: 108620
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

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

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