+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-20626 | |||||||||||||||||||||||||||
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Title | AAV8 Baculovirus-Sf9 produced, full capsid | |||||||||||||||||||||||||||
Map data | AAV8 full capsid | |||||||||||||||||||||||||||
Sample |
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Keywords | Adeno-associated virus / AAV / gene therapy vector / post translational modification / capsid / VIRUS | |||||||||||||||||||||||||||
Function / homology | Phospholipase 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 species | Adeno-associated virus - 8 | |||||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.6 Å | |||||||||||||||||||||||||||
Authors | Paulk NK / Poweleit N | |||||||||||||||||||||||||||
Funding support | United States, 8 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_20626.map.gz | 474.2 MB | EMDB map data format | |
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Header (meta data) | emd-20626-v30.xml emd-20626.xml | 19.4 KB 19.4 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_20626_fsc.xml | 23.3 KB | Display | FSC data file |
Images | emd_20626.png | 139.2 KB | ||
Filedesc metadata | emd-20626.cif.gz | 6.2 KB | ||
Others | emd_20626_half_map_1.map.gz emd_20626_half_map_2.map.gz | 46.6 MB 46.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-20626 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-20626 | HTTPS FTP |
-Validation report
Summary document | emd_20626_validation.pdf.gz | 989.2 KB | Display | EMDB validaton report |
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Full document | emd_20626_full_validation.pdf.gz | 988.8 KB | Display | |
Data in XML | emd_20626_validation.xml.gz | 26.6 KB | Display | |
Data in CIF | emd_20626_validation.cif.gz | 35.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20626 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20626 | HTTPS FTP |
-Related structure data
Related structure data | 6u2vMC 6pwaC 6u20C 6ubmC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_20626.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | AAV8 full capsid | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.07 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: AAV8 full capsid
File | emd_20626_half_map_1.map | ||||||||||||
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Annotation | AAV8 full capsid | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: AAV8 full capsid
File | emd_20626_half_map_2.map | ||||||||||||
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Annotation | AAV8 full capsid | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Adeno-associated virus - 8
Entire | Name: Adeno-associated virus - 8 |
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Components |
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-Supramolecule #1: Adeno-associated virus - 8
Supramolecule | Name: Adeno-associated virus - 8 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all Details: Recombinant AAV8 vector, produced in suspension Spodoptera frugiperda (Sf9) cells infected with live baculovirus expressing AAV components. 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
Macromolecule | Name: Capsid protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Adeno-associated virus - 8 |
Molecular weight | Theoretical: 58.528367 KDa |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.4 Component:
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Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 395 / Average exposure time: 6.0 sec. / Average electron dose: 66.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
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Output model | PDB-6u2v: |