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- EMDB-46741: AAV8 in complex with the AAVX affinity ligand -

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Basic information

Entry
Database: EMDB / ID: EMD-46741
TitleAAV8 in complex with the AAVX affinity ligand
Map data
Sample
  • Complex: AAV8 capsid in complex with AAVX
    • Protein or peptide: Capsid protein
    • Protein or peptide: AAVX affinity ligand
KeywordsParvoviridae / AAV vector / capsid / Adeno-associated virus / VIRUS / AAVX
Function / homology
Function and homology information


T=1 icosahedral viral capsid / nucleotide binding / structural molecule activity
Similarity search - Function
Phospholipase A2-like domain / Phospholipase A2-like domain / Parvovirus coat protein VP2 / Parvovirus coat protein VP1/VP2 / Parvovirus coat protein VP2 / Capsid/spike protein, ssDNA virus
Similarity search - Domain/homology
Biological speciesadeno-associated virus 8 / Camelidae (mammal)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.31 Å
AuthorsMietzsch M / McKenna R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM082946 United States
CitationJournal: Mol Ther Methods Clin Dev / Year: 2024
Title: Structural characterization and epitope mapping of the AAVX affinity purification ligand.
Authors: Mario Mietzsch / Manasi Kamat / Kari Basso / Paul Chipman / Juha T Huiskonen / Robert McKenna /
Abstract: The application of adeno-associated virus (AAV) vectors in human gene therapies requires reproducible and homogeneous preparations for clinical efficacy and safety. For the AAV production process, ...The application of adeno-associated virus (AAV) vectors in human gene therapies requires reproducible and homogeneous preparations for clinical efficacy and safety. For the AAV production process, often scalable affinity chromatography columns are utilized, such as the POROS CaptureSelect AAVX affinity resin, during downstream processing to ensure highly purified AAV vectors. The AAVX ligand is based on a camelid single-domain antibody capturing a wide range of recombinant AAV capsids. Described here is the identification of the AAV8 capsid epitope to AAVX at 2.3 Å resolution using cryo-electron microscopy. The ligand binds near the 5-fold axis of the capsid in a similar manner to the previously characterized AVB affinity ligand but does not conform to the capsid's icosahedral symmetry. The cross-reactivity of AAVX to other AAV capsids is achieved by primarily interacting with the peptide backbone of the AAV capsid's structurally conserved DE and HI loops. These observations will guide AAV capsid engineering efforts to retain the ability of future recombinant capsid designs to be purified using antibody-based affinity ligands.
History
DepositionAug 25, 2024-
Header (metadata) releaseNov 27, 2024-
Map releaseNov 27, 2024-
UpdateJan 1, 2025-
Current statusJan 1, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46741.png

Downloads & links

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Map

FileDownload / File: emd_46741.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.84 Å/pix.
x 500 pix.
= 420. Å		0.84 Å/pix.
x 500 pix.
= 420. Å		0.84 Å/pix.
x 500 pix.
= 420. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.4
Minimum - Maximum-8.408751000000001 - 17.458186999999999
Average (Standard dev.)-0.000000003857333 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-250-250-250
Dimensions500500500
Spacing500500500
CellA=B=C: 420.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_46741_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_46741_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : AAV8 capsid in complex with AAVX

EntireName: AAV8 capsid in complex with AAVX
Components
  • Complex: AAV8 capsid in complex with AAVX
    • Protein or peptide: Capsid protein
    • Protein or peptide: AAVX affinity ligand

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Supramolecule #1: AAV8 capsid in complex with AAVX

SupramoleculeName: AAV8 capsid in complex with AAVX / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: adeno-associated virus 8

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Macromolecule #1: Capsid protein

MacromoleculeName: Capsid protein / type: protein_or_peptide / ID: 1 / Number of copies: 60 / Enantiomer: LEVO
Source (natural)Organism: adeno-associated virus 8
Molecular weightTheoretical: 59.943891 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAAGGGAPMA DNNEGADGVG SSSGNWHCDS TWLGDRVITT STRTWALPTY NNHLYKQISN GTSGGATNDN TYFGYSTPWG YFDFNRFHC HFSPRDWQRL INNNWGFRPK RLSFKLFNIQ VKEVTQNEGT KTIANNLTST IQVFTDSEYQ LPYVLGSAHQ G CLPPFPAD ...String:
MAAGGGAPMA DNNEGADGVG SSSGNWHCDS TWLGDRVITT STRTWALPTY NNHLYKQISN GTSGGATNDN TYFGYSTPWG YFDFNRFHC HFSPRDWQRL INNNWGFRPK RLSFKLFNIQ VKEVTQNEGT KTIANNLTST IQVFTDSEYQ LPYVLGSAHQ G CLPPFPAD VFMIPQYGYL TLNNGSQAVG RSSFYCLEYF PSQMLRTGNN FQFTYTFEDV PFHSSYAHSQ SLDRLMNPLI DQ YLYYLSR TQTTGGTANT QTLGFSQGGP NTMANQAKNW LPGPCYRQQR VSTTTGQNNN SNFAWTAGTK YHLNGRNSLA NPG IAMATH KDDEERFFPS NGILIFGKQN AARDNADYSD VMLTSEEEIK TTNPVATEEY GIVADNLQQQ NTAPQIGTVN SQGA LPGMV WQNRDVYLQG PIWAKIPHTD GNFHPSPLMG GFGLKHPPPQ ILIKNTPVPA DPPTTFNQSK LNSFITQYST GQVSV EIEW ELQKENSKRW NPEIQYTSNY YKSTSVDFAV NTEGVYSEPR PIGTRYLTRN L

UniProtKB: Capsid protein

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Macromolecule #2: AAVX affinity ligand

MacromoleculeName: AAVX affinity ligand / type: protein_or_peptide / ID: 2 / Number of copies: 60 / Enantiomer: LEVO
Source (natural)Organism: Camelidae (mammal)
Molecular weightTheoretical: 13.630024 KDa
Recombinant expressionOrganism: unidentified (others)
SequenceString:
QVQIQESGGG IVQAGGSLRL SCAASGRTHG MYAMGWFRQA PGKEREFVAV QDITASNTHY SSAVKGRFTL SRDNAKNTAY LQMNNLKPE DTAVYYCAAG PTLMSGSYNS ARDYDYWGQG TQVTVSS

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.31 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 92713
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: PROJECTION MATCHING

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