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- EMDB-15286: Full AAV3B-VP1KO virion -

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Basic information

Entry
Database: EMDB / ID: EMD-15286
TitleFull AAV3B-VP1KO virion
Map dataSharpened cryo-EM map of the full AAV3B-VP1KO particle
Sample
  • Virus: Adeno-associated virus - 3
    • Protein or peptide: Capsid protein VP1
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) / Adeno-associated virus - 3
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 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-
UpdateNov 23, 2022-
Current statusNov 23, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15286.png

Downloads & links

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Map

FileDownload / File: emd_15286.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM map of the full AAV3B-VP1KO 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: 0.06
Minimum - Maximum-0.15927657 - 0.32582143
Average (Standard dev.)0.0012129318 (±0.019890256)
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_15286_additional_1.map
AnnotationReconstruction of the final iteration of the 3D auto refinement of the full AAV3B-VP1KO 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_15286_half_map_1.map
AnnotationUnfiltered half-1 map of the 3D auto refinement of the full AAV3B-VP1KO 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_15286_half_map_2.map
AnnotationUnfiltered half-2 map of the 3D auto refinement of the full AAV3B-VP1KO 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
    • Protein or peptide: Capsid protein VP1

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Supramolecule #1: Adeno-associated virus - 3

SupramoleculeName: Adeno-associated virus - 3 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: For the recombinant production of the VP1KO 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)
Host systemOrganism: Homo sapiens (human)
Virus shellShell ID: 1 / Diameter: 260.0 Å / T number (triangulation number): 1

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Macromolecule #1: Capsid protein VP1

MacromoleculeName: Capsid protein VP1 / type: protein_or_peptide / ID: 1
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.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 66.728281 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: TAPGKKRPVD QSPQEPDSSS GVGKSGKQPA RKRLNFGQTG DSESVPDPQP LGEPPAAPTS LGSNTMASGG GAPMADNNEG ADGVGNSSG NWHCDSQWLG DRVITTSTRT WALPTYNNHL YKQISSQSGA SNDNHYFGYS TPWGYFDFNR FHCHFSPRDW Q RLINNNWG ...String:
TAPGKKRPVD QSPQEPDSSS GVGKSGKQPA RKRLNFGQTG DSESVPDPQP LGEPPAAPTS LGSNTMASGG GAPMADNNEG ADGVGNSSG NWHCDSQWLG DRVITTSTRT WALPTYNNHL YKQISSQSGA SNDNHYFGYS TPWGYFDFNR FHCHFSPRDW Q RLINNNWG FRPKKLSFKL FNIQVKEVTQ NDGTTTIANN LTSTVQVFTD SEYQLPYVLG SAHQGCLPPF PADVFMVPQY GY LTLNNGS QAVGRSSFYC LEYFPSQMLR TGNNFQFSYT FEDVPFHSSY AHSQSLDRLM NPLIDQYLYY LNRTQGTTSG TTN QSRLLF SQAGPQSMSL QARNWLPGPC YRQQRLSKTA NDNNNSNFPW TAASKYHLNG RDSLVNPGPA MASHKDDEEK FFPM HGNLI FGKEGTTASN AELDNVMITD EEEIRTTNPV ATEQYGTVAN NLQSSNTAPT TRTVNDQGAL PGMVWQDRDV YLQGP IWAK IPHTDGHFHP SPLMGGFGLK HPPPQIMIKN TPVPANPPTT FSPAKFASFI TQYSTGQVSV EIEWELQKEN SKRWNP EIQ YTSNYNKSVN VDFTVDTNGV YSEPRPIGTR YLTRNL

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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..
DetailsVP1KO 4.23E+13 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: 401717
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.13)
Startup modelType of model: EMDB MAP
EMDB ID:

20624

Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 340947
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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Atomic model buiding 1

Initial modelPDB ID:

3kic

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8a9u:
Full AAV3B-VP1KO virion

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