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- PDB-8a9u: Full AAV3B-VP1KO virion -

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Basic information

Entry
Database: PDB / ID: 8a9u
TitleFull AAV3B-VP1KO virion
ComponentsCapsid protein VP1
KeywordsVIRUS / Adeno-Associated Virus / AAV / AAV3B / AAV serotype 3B / Full / VP1KO
Function / homologyPhospholipase 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 / T=1 icosahedral viral capsid / structural molecule activity / Capsid protein VP1
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsArriaga, I. / Abrescia, N.G.A.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Hum Gene Ther / Year: 2022
Title: Cellular and Structural Characterization of VP1 and VP2 Knockout Mutants of AAV3B Serotype and Implications for AAV Manufacturing.
Authors: Iker Arriaga / Aitor Navarro / Amaia Etxabe / César Trigueros / R Jude Samulski / Philippe Moullier / Achille François / Nicola G A Abrescia /
Abstract: AAV virion biology is still lacking a complete understanding of the role that the various structural subunits (VP1, 2, and 3) play in virus assembly, infectivity, and therapeutic delivery for ...AAV virion biology is still lacking a complete understanding of the role that the various structural subunits (VP1, 2, and 3) play in virus assembly, infectivity, and therapeutic delivery for clinical indications. In this study, we focus on the less studied adeno-associated virus AAV3B and generate a collection of AAV plasmid substrates that assemble virion particles deficient specifically in VP1, VP2, or VP1 and 2 structural subunits. Using a collection of biological and structural assays, we observed that virions devoid of VP1, VP2, or VP1 and 2 efficiently assembled virion particles, indistinguishable by cryoelectron microscopy (cryo-EM) from that of wild type (WT), but unique in virion transduction (WT > VP2 > VP1 > VP1 and 2 mutants). We also observed that the missing structural subunit was mostly compensated by additional VP3 protomers in the formed virion particle. Using cryo-EM analysis, virions fell into three classes, namely full, empty, and partially filled, based on comparison of density values within the capsid. Further, we characterize virions described as "broken" or "disassembled" particles, and provide structural information that supports the particle dissolution occurring through the two-fold symmetry sites. Finally, we highlight the unique value of employing cryo-EM as an essential tool for release criteria with respect to AAV manufacturing.
History
DepositionJun 29, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
0: Capsid protein VP1


Theoretical massNumber of molelcules
Total (without water)66,7281
Polymers66,7281
Non-polymers00
Water00
1
0: Capsid protein VP1
x 60


Theoretical massNumber of molelcules
Total (without water)4,003,69760
Polymers4,003,69760
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59

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Components

#1: Protein Capsid protein VP1


Mass: 66728.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: For pXR3b-VP1KO, the ATG start codon of VP1 was changed to TGA, and the GAT codon of Asp4 was changed to GAC to remove an alternative ATG. Thus this sample only has VP2 and VP3.
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pXR3b / Details (production host): AAV2 rep and AAV3B cap genes / Cell line (production host): Pro10TM / Production host: Homo sapiens (human) / References: UniProt: O56139

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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 - 3 / Type: VIRUS
Details: For the recombinant production of the VP1KO virion, we used the Pro10TM cell line.
Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Adeno-associated virus - 3 / Strain: 3B
Source (recombinant)Organism: Homo sapiens (human)
Details of virusEmpty: YES / Enveloped: NO / Isolate: SEROTYPE / Type: VIRION
Natural hostOrganism: Homo sapiens
Virus shellDiameter: 260 nm / Triangulation number (T number): 1
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: VP1KO 4.23E+13 vg/ml
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 283 K
Details: Parameters: 2 seconds of blot time, -2 offset value, after 45 seconds of incubation. Quantifoil Cu 300-mesh R1.2/1.3 holey-carbon grids with an extra layer of carbon on top added in-house.

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 2384

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
2RELION3.1particle selection
5CTFFIND4.1.13CTF correction
8PHENIXmodel fitting
10PHENIXmodel refinement
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 401717
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 340947 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 3KIC

3kic

RefinementHighest resolution: 3.1 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00325668
ELECTRON MICROSCOPYf_angle_d0.52935040
ELECTRON MICROSCOPYf_dihedral_angle_d4.6513396
ELECTRON MICROSCOPYf_chiral_restr0.043642
ELECTRON MICROSCOPYf_plane_restr0.0044668

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