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- EMDB-15287: Broken-(Dis)assembled AAV3B-VP2KO particle -

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Basic information

Entry
Database: EMDB / ID: EMD-15287
TitleBroken-(Dis)assembled AAV3B-VP2KO particle
Map dataSharpened cryo-EM map of the broken-(dis)assembled AAV3B-VP2KO particle
Sample
  • Virus: Adeno-associated virus - 3
KeywordsAdeno-Associated Virus / AAV / AAV3B / AAV serotype 3B / Broken / Disassembled / VIRUS
Biological speciesAdeno-associated virus - 3
Methodsingle particle reconstruction / cryo EM / Resolution: 25.0 Å
AuthorsArriaga I / Abrescia NGA
Funding support1 items
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, 2022-
Header (metadata) releaseSep 21, 2022-
Map releaseSep 21, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15287.png

Downloads & links

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Map

FileDownload / File: emd_15287.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM map of the broken-(dis)assembled AAV3B-VP2KO particle
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.56 Å/pix.
x 300 pix.
= 466.8 Å		1.56 Å/pix.
x 300 pix.
= 466.8 Å		1.56 Å/pix.
x 300 pix.
= 466.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.556 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.026721945 - 0.06359494
Average (Standard dev.)0.00031426473 (±0.0077212527)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 466.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Reconstruction of the final iteration of the 3D...

Fileemd_15287_additional_1.map
AnnotationReconstruction of the final iteration of the 3D auto refinement of the broken-(dis)assembled AAV3B-VP2KO particle
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half-1 map of the 3D auto refinement...

Fileemd_15287_half_map_1.map
AnnotationUnfiltered half-1 map of the 3D auto refinement of the broken-(dis)assembled AAV3B-VP2KO particle
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half-2 map of the 3D auto refinement...

Fileemd_15287_half_map_2.map
AnnotationUnfiltered half-2 map of the 3D auto refinement of the broken-(dis)assembled AAV3B-VP2KO particle
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Adeno-associated virus - 3

EntireName: Adeno-associated virus - 3
Components
  • Virus: Adeno-associated virus - 3

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Supramolecule #1: Adeno-associated virus - 3

SupramoleculeName: Adeno-associated virus - 3 / type: virus / ID: 1 / Parent: 0
Details: For the recombinant production of the virion, we used the Pro10TM cell line.
NCBI-ID: 46350 / Sci species name: Adeno-associated virus - 3 / Sci species strain: 3B / Virus type: VIRION / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human)
Virus shellShell ID: 1 / Diameter: 260.0 Å / T number (triangulation number): 1

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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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK III
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..
Details9.40E+12 vg/ml

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Number real images: 2384 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 92000

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Image processing

Particle selectionNumber selected: 202784
Startup modelType of model: EMDB MAP
EMDB ID:

20624

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 1201
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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