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- PDB-6ubm: AAV8 Baculovirus-Sf9 produced, empty capsid -

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Basic information

Entry
Database: PDB / ID: 6ubm
TitleAAV8 Baculovirus-Sf9 produced, empty capsid
ComponentsCapsid proteinCapsid
KeywordsVIRUS / Adeno-associated virus / AAV / gene therapy vector / post translational modification / capsid
Function / homology
Function and homology information


T=1 icosahedral viral capsid / structural molecule activity
Similarity search - Function
Empty Capsid Viral Protein 2 / Parvovirus coat protein VP1/VP2 / 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 / Beta Complex / Mainly Beta
Similarity search - Domain/homology
Biological speciesAdeno-associated virus - 8
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsPaulk, N.K. / Poweleit, N.
Funding support United States, 8items
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
DepositionSep 12, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 3, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 14, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Mar 20, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / pdbx_struct_oper_list
Item: _citation.country / _database_2.pdbx_DOI ..._citation.country / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Capsid protein


Theoretical massNumber of molelcules
Total (without water)58,5281
Polymers58,5281
Non-polymers00
Water0
1
A: Capsid protein
x 60


Theoretical massNumber of molelcules
Total (without water)3,511,70260
Polymers3,511,70260
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: Capsid protein
x 5


  • icosahedral pentamer
  • 293 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)292,6425
Polymers292,6425
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Capsid protein
x 6


  • icosahedral 23 hexamer
  • 351 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)351,1706
Polymers351,1706
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein Capsid protein / Capsid


Mass: 58528.367 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus - 8 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8JQF8

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Adeno-associated virus - 8 / Type: VIRUS
Details: Recombinant AAV8 vector, produced in suspension Spodoptera frugiperda (Sf9) cells infected with live baculovirus expressing AAV components.
Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Adeno-associated virus - 8
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Cell: Sf9
Details of virusEmpty: YES / Enveloped: NO / Isolate: SEROTYPE / Type: VIRION
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1317 mMsodium chlorideNaClSodium chloride1
20.001 %Lutrol1
32.67 mMPotassium ChlorideKCl1
41.5 mMPotassium Phosphate MonobasicKH2PO41
58.1 mMSodium Phosphate DibasicNa2HPO41
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 66 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 395
Image scansMovie frames/image: 30

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Processing

EM software
IDNameCategory
2EPUimage acquisition
4cisTEMCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cisTEMinitial Euler assignment
11cisTEMfinal Euler assignment
12cisTEMclassification
13cisTEM3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2395 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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