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- EMDB-54195: AAV8 capsid in complex with Rep40 and ADP -

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Basic information

Entry
Database: EMDB / ID: EMD-54195
TitleAAV8 capsid in complex with Rep40 and ADP
Map data
Sample
  • Virus: adeno-associated virus 8
    • Complex: AAV2 Rep40
      • Protein or peptide: Protein Rep40
    • Protein or peptide: Capsid protein
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsAdeno-associated virus / AAV / AAA+ ATPase / VIRUS
Function / homology
Function and homology information


T=1 icosahedral viral capsid / viral genome replication / DNA helicase activity / DNA helicase / DNA replication / nucleotide binding / host cell nucleus / structural molecule activity / 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. / Phospholipase A2-like domain / Phospholipase A2-like domain / Parvovirus coat protein VP2 / Parvovirus coat protein VP1/VP2 / Parvovirus coat protein VP1/VP2 / Capsid/spike protein, ssDNA virus / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Protein Rep40 / Capsid protein
Similarity search - Component
Biological speciesadeno-associated virus 2 / adeno-associated virus 8
Methodsingle particle reconstruction / cryo EM / Resolution: 1.88 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
DepositionJun 28, 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_54195.png

Downloads & links

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Map

FileDownload / File: emd_54195.map.gz / Format: CCP4 / Size: 600.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 540 pix.
= 502.74 Å		0.93 Å/pix.
x 540 pix.
= 502.74 Å		0.93 Å/pix.
x 540 pix.
= 502.74 Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.931 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0608
Minimum - Maximum-0.13320364 - 0.45175856
Average (Standard dev.)0.0027765885 (±0.021371868)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions540540540
Spacing540540540
CellA=B=C: 502.74 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_54195_additional_1.map
Projections & Slices
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Half map: #1

Fileemd_54195_half_map_1.map
Projections & Slices
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Histogram

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Half map: #2

Fileemd_54195_half_map_2.map
Projections & Slices
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Sample components

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Entire : adeno-associated virus 8

EntireName: adeno-associated virus 8
Components
  • Virus: adeno-associated virus 8
    • Complex: AAV2 Rep40
      • Protein or peptide: Protein Rep40
    • Protein or peptide: Capsid protein
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: adeno-associated virus 8

SupramoleculeName: adeno-associated virus 8 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / NCBI-ID: 202813 / Sci species name: adeno-associated virus 8 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human)
Virus shellShell ID: 1 / Name: Virus protein (VP) / Diameter: 210.0 Å / T number (triangulation number): 1

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Supramolecule #2: AAV2 Rep40

SupramoleculeName: AAV2 Rep40 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: Segments of capsid-bound Rep40 aligned with the capsid icosahedral symmetry
Source (natural)Organism: adeno-associated virus 2

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Macromolecule #1: Protein Rep40

MacromoleculeName: Protein Rep40 / type: protein_or_peptide / ID: 1 / Details: Latch-WTAAV2Rep40-TrB-3xFLAG-TEV-6xHis / Number of copies: 1 / 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 #2: Capsid protein

MacromoleculeName: Capsid protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: adeno-associated virus 8
Molecular weightTheoretical: 81.833047 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAADGYLPDW LEDNLSEGIR EWWALKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLQAGDNP YLRYNHADAE FQERLQEDTS FGGNLGRAVF QAKKRVLEPL GLVEEGAKTA PGKKRPVEPS PQRSPDSSTG I GKKGQQPA ...String:
MAADGYLPDW LEDNLSEGIR EWWALKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLQAGDNP YLRYNHADAE FQERLQEDTS FGGNLGRAVF QAKKRVLEPL GLVEEGAKTA PGKKRPVEPS PQRSPDSSTG I GKKGQQPA RKRLNFGQTG DSESVPDPQP LGEPPAAPSG VGPNTMAAGG GAPMADNNEG ADGVGSSSGN WHCDSTWLGD RV ITTSTRT WALPTYNNHL YKQISNGTSG GATNDNTYFG YSTPWGYFDF NRFHCHFSPR DWQRLINNNW GFRPKRLSFK LFN IQVKEV TQNEGTKTIA NNLTSTIQVF TDSEYQLPYV LGSAHQGCLP PFPADVFMIP QYGYLTLNNG SQAVGRSSFY CLEY FPSQM LRTGNNFQFT YTFEDVPFHS SYAHSQSLDR LMNPLIDQYL YYLSRTQTTG GTANTQTLGF SQGGPNTMAN QAKNW LPGP CYRQQRVSTT TGQNNNSNFA WTAGTKYHLN GRNSLANPGI AMATHKDDEE RFFPSNGILI FGKQNAARDN ADYSDV MLT SEEEIKTTNP VATEEYGIVA DNLQQQNTAP QIGTVNSQGA LPGMVWQNRD VYLQGPIWAK IPHTDGNFHP SPLMGGF GL KHPPPQILIK NTPVPADPPT TFNQSKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTS VDFAVNTE G VYSEPRPIGT RYLTRNL

UniProtKB: Capsid protein

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / 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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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 217 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 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: 130000
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: 1.88 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Number images used: 125371
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 modelPDB ID:

6v12


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-9rrs:
AAV8 capsid in complex with Rep40 and ADP

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