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- EMDB-20625: AAV3B genome containing -

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Basic information

Entry
Database: EMDB / ID: EMD-20625
TitleAAV3B genome containing
Map dataGenome containing AAV3B
SampleParvoviridae != Adeno-associated virus 3B

Parvoviridae

  • Virus: Adeno-associated virus 3B
    • Protein or peptide: AAV3B VP1
Biological speciesHomo sapiens (human) / Adeno-associated virus 3B
Methodsingle particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsSubramanian S / Hafenstein S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute1S10OD011986-01A1 United States
CitationJournal: Hum Gene Ther / Year: 2019
Title: Filling Adeno-Associated Virus Capsids: Estimating Success by Cryo-Electron Microscopy.
Authors: Suriyasri Subramanian / Anna C Maurer / Carol M Bator / Alexander M Makhov / James F Conway / Kevin B Turner / James H Marden / Luk H Vandenberghe / Susan L Hafenstein /
Abstract: Adeno-associated viruses (AAVs) have been employed successfully as gene therapy vectors in treating various genetic diseases for almost two decades. However, transgene packaging is usually imperfect, ...Adeno-associated viruses (AAVs) have been employed successfully as gene therapy vectors in treating various genetic diseases for almost two decades. However, transgene packaging is usually imperfect, and developing a rapid and accurate method for measuring the proportion of DNA encapsidation is an important step for improving the downstream process of large scale vector production. In this study, we used two-dimensional class averages and three-dimensional classes, intermediate outputs in the single particle cryo-electron microscopy (cryo-EM) image reconstruction pipeline, to determine the proportion of DNA-packaged and empty capsid populations. Two different preparations of AAV3 were analyzed to estimate the minimum number of particles required to be sampled by cryo-EM in order for robust calculation of the proportion of the full versus empty capsids in any given sample. Cost analysis applied to the minimum amount of data required for a valid ratio suggests that cryo-EM is an effective approach to analyze vector preparations.
History
DepositionAug 20, 2019-
Header (metadata) releaseSep 4, 2019-
Map releaseOct 2, 2019-
UpdateJan 1, 2020-
Current statusJan 1, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20625.png

Downloads & links

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Map

FileDownload / File: emd_20625.map.gz / Format: CCP4 / Size: 193.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGenome containing AAV3B
Voxel sizeX=Y=Z: 1.136 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.23371518 - 0.40958893
Average (Standard dev.)0.001722293 (±0.021821838)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions370370370
Spacing370370370
CellA=B=C: 420.32 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1361.1361.136
M x/y/z370370370
origin x/y/z0.0000.0000.000
length x/y/z420.320420.320420.320
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ720720720
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS370370370
D min/max/mean-0.2340.4100.002

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Supplemental data

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Sample components

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Entire : Parvoviridae

EntireName: Parvoviridae (virus)
Components
  • Virus: Adeno-associated virus 3B
    • Protein or peptide: AAV3B VP1

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Supramolecule #1: Adeno-associated virus 3B

SupramoleculeName: Adeno-associated virus 3B / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 68742 / Sci species name: Adeno-associated virus 3B / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Mus spretus (western wild mouse)
Host systemOrganism: Homo sapiens (human) / Recombinant cell: HEK
Virus shellShell ID: 1 / Diameter: 250.0 Å / T number (triangulation number): 1

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Macromolecule #1: AAV3B VP1

MacromoleculeName: AAV3B VP1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAADGYLPDW LEDNLSEGIR EWWALKPGVP QPKANQQHQD NRRGLVLPGY KYLGPGNGLD KGEPVNEAD AAALEHDKAY DQQLKAGDNP YLKYNHADAE FQERLQEDTS FGGNLGRAVF Q AKKRILEP LGLVEEAAKT APGKKRPVDQ SPQEPDSSSG VGKSGKQPAR ...String:
MAADGYLPDW LEDNLSEGIR EWWALKPGVP QPKANQQHQD NRRGLVLPGY KYLGPGNGLD KGEPVNEAD AAALEHDKAY DQQLKAGDNP YLKYNHADAE FQERLQEDTS FGGNLGRAVF Q AKKRILEP LGLVEEAAKT APGKKRPVDQ SPQEPDSSSG VGKSGKQPAR KRLNFGQTGD SE SVPDPQP LGEPPAAPTS LGSNTMASGG GAPMADNNEG ADGVGNSSGN WHCDSQWLGD RVI TTSTRT WALPTYNNHL YKQISSQSGA SNDNHYFGYS TPWGYFDFNR FHCHFSPRDW QRLI NNNWG FRPKKLSFKL FNIQVKEVTQ NDGTTTIANN LTSTVQVFTD SEYQLPYVLG SAHQG CLPP FPADVFMVPQ YGYLTLNNGS QAVGRSSFYC LEYFPSQMLR TGNNFQFSYT FEDVPF HSS YAHSQSLDRL MNPLIDQYLY YLNRTQGTTS GTTNQSRLLF SQAGPQSMSL QARNWLP GP CYRQQRLSKT ANDNNNSNFP WTAASKYHLN GRDSLVNPGP AMASHKDDEE KFFPMHGN L IFGKEGTTAS NAELDNVMIT DEEEIRTTNP VATEQYGTVA NNLQSSNTAP TTRTVNDQG ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT TFSPAKFAS FITQYSTGQV SVEIEWELQK ENSKRWNPEI QYTSNYNKSV NVDFTVDTNG V YSEPRPIG TRYLTRNL

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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
Component:
ConcentrationFormulaName
137.0 mMNaClSodium chloridesodium chloride
1.7 mMKClpotassium chloride
10.0 mMNa2HPO4sodium phosphate dibasic
1.8 mMKH2PO4potassium phosphate monobasic

Details: Phosphate buffered saline pH 7.4
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 2082 / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 132631
Details: Manual picking was used to handpick 1000 particles which were used as template to then autopick
CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 31488
FSC plot (resolution estimation)

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