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- PDB-9dc3: AAV8 in complex with the AAVX affinity ligand -

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Basic information

Entry
Database: PDB / ID: 9dc3
TitleAAV8 in complex with the AAVX affinity ligand
Components
  • AAVX affinity ligand
  • Capsid protein
KeywordsVIRUS / Parvoviridae / AAV vector / capsid / Adeno-associated 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 VP1/VP2 / Capsid/spike protein, ssDNA virus
Similarity search - Domain/homology
Biological speciesadeno-associated virus 8
Camelidae (mammal)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.31 Å
AuthorsMietzsch, M. / McKenna, R.
Funding support United States, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 27, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 1, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Capsid protein
B: Capsid protein
C: Capsid protein
D: Capsid protein
E: Capsid protein
F: Capsid protein
G: Capsid protein
H: Capsid protein
I: Capsid protein
J: Capsid protein
K: Capsid protein
L: Capsid protein
M: Capsid protein
N: Capsid protein
O: Capsid protein
P: Capsid protein
Q: Capsid protein
R: Capsid protein
S: Capsid protein
T: Capsid protein
U: Capsid protein
V: Capsid protein
W: Capsid protein
X: Capsid protein
Y: Capsid protein
Z: Capsid protein
a: Capsid protein
b: Capsid protein
c: Capsid protein
d: Capsid protein
e: Capsid protein
f: Capsid protein
g: Capsid protein
h: Capsid protein
i: Capsid protein
j: Capsid protein
k: Capsid protein
l: Capsid protein
m: Capsid protein
n: Capsid protein
o: Capsid protein
p: Capsid protein
q: Capsid protein
r: Capsid protein
s: Capsid protein
t: Capsid protein
u: Capsid protein
v: Capsid protein
w: Capsid protein
x: Capsid protein
y: Capsid protein
z: Capsid protein
1: Capsid protein
2: Capsid protein
3: Capsid protein
4: Capsid protein
5: Capsid protein
6: Capsid protein
7: Capsid protein
8: Capsid protein
A2: AAVX affinity ligand
B2: AAVX affinity ligand
C2: AAVX affinity ligand
D2: AAVX affinity ligand
E2: AAVX affinity ligand
F2: AAVX affinity ligand
G2: AAVX affinity ligand
H2: AAVX affinity ligand
I2: AAVX affinity ligand
J2: AAVX affinity ligand
K2: AAVX affinity ligand
L2: AAVX affinity ligand
M2: AAVX affinity ligand
N2: AAVX affinity ligand
O2: AAVX affinity ligand
P2: AAVX affinity ligand
Q2: AAVX affinity ligand
R2: AAVX affinity ligand
S2: AAVX affinity ligand
T2: AAVX affinity ligand
U2: AAVX affinity ligand
V2: AAVX affinity ligand
W2: AAVX affinity ligand
X2: AAVX affinity ligand
Y2: AAVX affinity ligand
Z2: AAVX affinity ligand
a2: AAVX affinity ligand
b2: AAVX affinity ligand
c2: AAVX affinity ligand
d2: AAVX affinity ligand
e2: AAVX affinity ligand
f2: AAVX affinity ligand
g2: AAVX affinity ligand
h2: AAVX affinity ligand
i2: AAVX affinity ligand
j2: AAVX affinity ligand
k2: AAVX affinity ligand
l2: AAVX affinity ligand
m2: AAVX affinity ligand
n2: AAVX affinity ligand
o2: AAVX affinity ligand
p2: AAVX affinity ligand
q2: AAVX affinity ligand
r2: AAVX affinity ligand
s2: AAVX affinity ligand
t2: AAVX affinity ligand
u2: AAVX affinity ligand
v2: AAVX affinity ligand
w2: AAVX affinity ligand
x2: AAVX affinity ligand
y2: AAVX affinity ligand
z2: AAVX affinity ligand
12: AAVX affinity ligand
22: AAVX affinity ligand
32: AAVX affinity ligand
42: AAVX affinity ligand
52: AAVX affinity ligand
62: AAVX affinity ligand
72: AAVX affinity ligand
82: AAVX affinity ligand


Theoretical massNumber of molelcules
Total (without water)4,414,435120
Polymers4,414,435120
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein ...
Capsid protein


Mass: 59943.891 Da / Num. of mol.: 60
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) adeno-associated virus 8 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q8JQF8
#2: Antibody ...
AAVX affinity ligand


Mass: 13630.024 Da / Num. of mol.: 60
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelidae (mammal) / Production host: unidentified (others)
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: AAV8 capsid in complex with AAVX / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: adeno-associated virus 8
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Details of virusEmpty: NO / Enveloped: NO / Isolate: SEROTYPE / Type: VIRION
Virus shellTriangulation number (T number): 1
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.10-2155_2155: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.31 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 92713 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.013313860
ELECTRON MICROSCOPYf_angle_d1.031427740
ELECTRON MICROSCOPYf_dihedral_angle_d10.032248580
ELECTRON MICROSCOPYf_chiral_restr0.0744700
ELECTRON MICROSCOPYf_plane_restr0.00756820

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