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- EMDB-54181: Consensus map of heptameric Rep40-dsDNA (ITR) in presence of ATPyS -

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Basic information

Entry
Database: EMDB / ID: EMD-54181
TitleConsensus map of heptameric Rep40-dsDNA (ITR) in presence of ATPyS
Map data
Sample
  • Complex: AAV2 Heptameric Rep40-dsDNA (ITR) in presence of ATPgS
KeywordsHelicase / Adeno-associated Virus / AAA+ ATPase / DNA BINDING PROTEIN
Biological speciesadeno-associated virus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 2.75 Å
AuthorsRouse SL / Bubeck D / Barritt JD / Xu V / Wake M
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Other government United Kingdom
Other private United Kingdom
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
DepositionJun 26, 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_54181.png

Downloads & links

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Map

FileDownload / File: emd_54181.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.0243
Minimum - Maximum-0.099436454 - 0.25145334
Average (Standard dev.)-0.00005458173 (±0.0051027583)
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: #2

Fileemd_54181_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_54181_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 Heptameric Rep40-dsDNA (ITR) in presence of ATPgS

EntireName: AAV2 Heptameric Rep40-dsDNA (ITR) in presence of ATPgS
Components
  • Complex: AAV2 Heptameric Rep40-dsDNA (ITR) in presence of ATPgS

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Supramolecule #1: AAV2 Heptameric Rep40-dsDNA (ITR) in presence of ATPgS

SupramoleculeName: AAV2 Heptameric Rep40-dsDNA (ITR) in presence of ATPgS
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Adeno-associated Virus 2 Replication protein 40 in complex with inverted-terminal-repeat DNA 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

Concentration0.8 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
200.0 mMNaClSodium Chloride
25.0 mM(HOCH2)*3CNH2Tris
1.0 mMC9H15O6PTCEP
1.0 %C3H8O3Glycerol
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
SoftwareName: EPU (ver. 3.8.1)
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average exposure time: 1.68 sec. / 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 correctionSoftware - Name: cryoSPARC (ver. 4.4.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.75 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Number images used: 137594
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
SoftwareName: ISOLDE (ver. 1.8)

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