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- PDB-7rl1: AAVrh.10-7x capsid -

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Basic information

Entry
Database: PDB / ID: 7rl1
TitleAAVrh.10-7x capsid
ComponentsCapsid protein VP1
KeywordsVIRUS / Icosahedral Capsid / AAVrh.10 / capsid engineering / Adeno-associated virus / Parvovirus / Gene Therapy
Function / homology
Function and homology information


T=1 icosahedral viral capsid / 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
2'-DEOXYADENOSINE-5'-MONOPHOSPHATE / 2'-DEOXYCYTIDINE-5'-MONOPHOSPHATE / Capsid protein VP1
Similarity search - Component
Biological speciesAdeno-associated virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.71 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: J Virol / Year: 2021
Title: Structural Study of Aavrh.10 Receptor and Antibody Interactions.
Authors: Mario Mietzsch / Jennifer C Yu / Jane Hsi / Paul Chipman / Felix Broecker / Zhang Fuming / Robert J Linhardt / Peter H Seeberger / Regine Heilbronn / Robert McKenna / Mavis Agbandje-McKenna /
Abstract: Recombinant adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, ...Recombinant adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, the AAV vectors have to circumvent potential preexisting neutralizing host antibodies and bind to the receptors of the target cells. Both of these aspects have not been structurally analyzed for AAVrh.10. Here, cryo-electron microscopy and three-dimensional image reconstruction were used to map the binding site of sulfated -acetyllactosamine (LacNAc; previously shown to bind AAVrh.10) and a series of four monoclonal antibodies (MAbs). LacNAc was found to bind to a pocket located on the side of the 3-fold capsid protrusion that is mostly conserved to AAV9 and equivalent to its galactose-binding site. As a result, AAVrh.10 was also shown to be able to bind to cell surface glycans with terminal galactose. For the antigenic characterization, it was observed that several anti-AAV8 MAbs cross-react with AAVrh.10. The binding sites of these antibodies were mapped to the 3-fold capsid protrusions. Based on these observations, the AAVrh.10 capsid surface was engineered to create variant capsids that escape these antibodies while maintaining infectivity. Gene therapy vectors based on adeno-associated virus rhesus isolate 10 (AAVrh.10) have been used in several clinical trials to treat monogenetic diseases. However, compared to other AAV serotypes little is known about receptor binding and antigenicity of the AAVrh.10 capsid. Particularly, preexisting neutralizing antibodies against capsids are an important challenge that can hamper treatment efficiency. This study addresses both topics and identifies critical regions of the AAVrh.10 capsid for receptor and antibody binding. The insights gained were utilized to generate AAVrh.10 variants capable of evading known neutralizing antibodies. The findings of this study could further aid the utilization of AAVrh.10 vectors in clinical trials and help the approval of the subsequent biologics.
History
DepositionJul 23, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 8, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 6, 2021Group: Database references / Structure summary / Category: citation / citation_author / pdbx_contact_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Nov 24, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Capsid protein VP1
B: Capsid protein VP1
C: Capsid protein VP1
D: Capsid protein VP1
E: Capsid protein VP1
F: Capsid protein VP1
G: Capsid protein VP1
H: Capsid protein VP1
I: Capsid protein VP1
J: Capsid protein VP1
K: Capsid protein VP1
L: Capsid protein VP1
M: Capsid protein VP1
N: Capsid protein VP1
O: Capsid protein VP1
P: Capsid protein VP1
Q: Capsid protein VP1
R: Capsid protein VP1
S: Capsid protein VP1
T: Capsid protein VP1
U: Capsid protein VP1
V: Capsid protein VP1
W: Capsid protein VP1
X: Capsid protein VP1
Y: Capsid protein VP1
Z: Capsid protein VP1
a: Capsid protein VP1
b: Capsid protein VP1
c: Capsid protein VP1
d: Capsid protein VP1
e: Capsid protein VP1
f: Capsid protein VP1
g: Capsid protein VP1
h: Capsid protein VP1
i: Capsid protein VP1
j: Capsid protein VP1
k: Capsid protein VP1
l: Capsid protein VP1
m: Capsid protein VP1
n: Capsid protein VP1
o: Capsid protein VP1
p: Capsid protein VP1
q: Capsid protein VP1
r: Capsid protein VP1
s: Capsid protein VP1
t: Capsid protein VP1
u: Capsid protein VP1
v: Capsid protein VP1
w: Capsid protein VP1
x: Capsid protein VP1
y: Capsid protein VP1
z: Capsid protein VP1
1: Capsid protein VP1
2: Capsid protein VP1
3: Capsid protein VP1
4: Capsid protein VP1
5: Capsid protein VP1
6: Capsid protein VP1
7: Capsid protein VP1
8: Capsid protein VP1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)3,537,707180
Polymers3,499,40260
Non-polymers38,305120
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Capsid protein VP1 /


Mass: 58323.359 Da / Num. of mol.: 60 / Fragment: UNP residues 219-738
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus / Gene: cap / Variant: rh.10-7x / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q6JC62
#2: Chemical...
ChemComp-DC / 2'-DEOXYCYTIDINE-5'-MONOPHOSPHATE / Deoxycytidine monophosphate


Type: DNA linking / Mass: 307.197 Da / Num. of mol.: 60 / Source method: obtained synthetically / Formula: C9H14N3O7P / Comment: dCMP*YM
#3: Chemical...
ChemComp-DA / 2'-DEOXYADENOSINE-5'-MONOPHOSPHATE / Deoxyadenosine monophosphate


Type: DNA linking / Mass: 331.222 Da / Num. of mol.: 60 / Source method: obtained synthetically / Formula: C10H14N5O6P / Comment: dAMP*YM

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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: Adeno-associated virus / Type: VIRUS / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Adeno-associated virus / Strain: rh.10-7x
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Details of virusEmpty: NO / Enveloped: NO / Isolate: OTHER / Type: VIRION
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 FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingElectron dose: 75 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.10-2155_2155: / Classification: refinement
EM software
IDNameCategory
2cisTEMimage acquisition
4cisTEMCTF correction
13cisTEM3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17861 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.009257100
ELECTRON MICROSCOPYf_angle_d0.822351060
ELECTRON MICROSCOPYf_dihedral_angle_d8.426202680
ELECTRON MICROSCOPYf_chiral_restr0.05536600
ELECTRON MICROSCOPYf_plane_restr0.00646260

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