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- EMDB-20502: AAV8 human HEK293-produced, full capsid -

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Basic information

Entry
Database: EMDB / ID: EMD-20502
TitleAAV8 human HEK293-produced, full capsid
Map dataAAV8 human HEK293-produced, full capsid
SampleAdeno-associated virus - 8 != Adeno-associated virus-8

Adeno-associated virus - 8

  • Virus: Adeno-associated virus-8
    • Protein or peptide: Capsid proteinCapsid
KeywordsAdeno-associated virus / AAV / gene therapy vector / post translational modification / capsid / VIRUS
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
Function and homology information
Biological speciesAdeno-associated virus - 8 / Adeno-associated virus-8
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsPaulk NK / Poweleit N
Funding support United States, 8 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)K01-DK107607 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)U01-HL145795 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)P01-HL51670 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)P30-DK54759 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P30-CA086862 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)U01-CA207702 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32-GM126663 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)T32-AI060537 United States
CitationJournal: Mol Ther Methods Clin Dev / Year: 2020
Title: Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors.
Authors: Neil G Rumachik / Stacy A Malaker / Nicole Poweleit / Lucy H Maynard / Christopher M Adams / Ryan D Leib / Giana Cirolia / Dennis Thomas / Susan Stamnes / Kathleen Holt / Patrick Sinn / ...Authors: Neil G Rumachik / Stacy A Malaker / Nicole Poweleit / Lucy H Maynard / Christopher M Adams / Ryan D Leib / Giana Cirolia / Dennis Thomas / Susan Stamnes / Kathleen Holt / Patrick Sinn / Andrew P May / Nicole K Paulk /
Abstract: Different approaches are used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection ...Different approaches are used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of () insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed a controlled comparative production analysis varying only the host cell species but maintaining all other parameters. We characterized differences with multiple analytical approaches: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM (transmission electron cryomicroscopy), denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and functional transduction assessments and , including in humanized liver mice. Using these approaches, we have made two major discoveries: (1) rAAV capsids have post-translational modifications (PTMs), including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms; and (2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data show that host cell protein impurities differ between platforms and can have their own PTMs, including potentially immunogenic N-linked glycans. Human-produced rAAVs are more potent than baculovirus- vectors in various cell types (p < 0.05-0.0001), in various mouse tissues (p < 0.03-0.0001), and in human liver (p < 0.005). These differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity, as well as cost considerations.
History
DepositionJul 22, 2019-
Header (metadata) releaseOct 2, 2019-
Map releaseJun 3, 2020-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6pwa
  • Surface level: 6
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6pwa
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20502.png

Downloads & links

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Map

FileDownload / File: emd_20502.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAAV8 human HEK293-produced, full capsid
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 6.0 / Movie #1: 6
Minimum - Maximum-31.820961 - 49.839550000000003
Average (Standard dev.)-0.024002682 (±2.1163585)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 547.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z547.840547.840547.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-31.82149.840-0.024

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Supplemental data

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Half map: AAV8 human HEK293-produced, full capsid

Fileemd_20502_half_map_1.map
AnnotationAAV8 human HEK293-produced, full capsid
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: AAV8 human HEK293-produced, full capsid

Fileemd_20502_half_map_2.map
AnnotationAAV8 human HEK293-produced, full capsid
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 - 8

EntireName: Adeno-associated virus - 8
Components
  • Virus: Adeno-associated virus-8
    • Protein or peptide: Capsid proteinCapsid

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Supramolecule #1: Adeno-associated virus-8

SupramoleculeName: Adeno-associated virus-8 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Recombinant AAV8 vector, produced in adherent human HEK293 cells purchased from ATCC.
NCBI-ID: 202813 / Sci species name: Adeno-associated virus-8 / Virus type: VIRION / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No

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Macromolecule #1: Capsid protein

MacromoleculeName: Capsid protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Adeno-associated virus - 8
Molecular weightTheoretical: 58.528367 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DGVGSSSGNW HCDSTWLGDR VITTSTRTWA LPTYNNHLYK QISNGTSGGA TNDNTYFGYS TPWGYFDFNR FHCHFSPRDW QRLINNNWG FRPKRLSFKL FNIQVKEVTQ NEGTKTIANN LTSTIQVFTD SEYQLPYVLG SAHQGCLPPF PADVFMIPQY G YLTLNNGS ...String:
DGVGSSSGNW HCDSTWLGDR VITTSTRTWA LPTYNNHLYK QISNGTSGGA TNDNTYFGYS TPWGYFDFNR FHCHFSPRDW QRLINNNWG FRPKRLSFKL FNIQVKEVTQ NEGTKTIANN LTSTIQVFTD SEYQLPYVLG SAHQGCLPPF PADVFMIPQY G YLTLNNGS QAVGRSSFYC LEYFPSQMLR TGNNFQFTYT FEDVPFHSSY AHSQSLDRLM NPLIDQYLYY LSRTQTTGGT AN TQTLGFS QGGPNTMANQ AKNWLPGPCY RQQRVSTTTG QNNNSNFAWT AGTKYHLNGR NSLANPGIAM ATHKDDEERF FPS NGILIF GKQNAARDNA DYSDVMLTSE EEIKTTNPVA TEEYGIVADN LQQQNTAPQI GTVNSQGALP GMVWQNRDVY LQGP IWAKI PHTDGNFHPS PLMGGFGLKH PPPQILIKNT PVPADPPTTF NQSKLNSFIT QYSTGQVSVE IEWELQKENS KRWNP EIQY TSNYYKSTSV DFAVNTEGVY SEPRPIGTRY LTRNL

UniProtKB: Capsid protein

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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
317.0 mMNaClSodium chloridesodium chloride
0.001 %Lutrol
2.67 mMKClPotassium Chloride
1.5 mMKH2PO4Potassium Phosphate Monobasic
8.1 mMNa2HPO4Sodium Phosphate Dibasic
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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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: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 1647 / Average exposure time: 6.0 sec. / Average electron dose: 66.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM
Final 3D classificationSoftware - Name: cisTEM
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 63471
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6pwa:
AAV8 human HEK293-produced, full capsid

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