+Open data
-Basic information
Entry | Database: PDB / ID: 7na6 | ||||||
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Title | Cryo-EM structure of AAV True Type | ||||||
Components | Capsid protein VP1 | ||||||
Keywords | VIRUS / Icosahedron / vector / therapeutic / beta-barrel | ||||||
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 VP1 Function and homology information | ||||||
Biological species | Adeno-associated virus | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å | ||||||
Authors | Bennett, A.D. / McKenna, R. | ||||||
Citation | Journal: J Struct Biol / Year: 2021 Title: Comparative structural, biophysical, and receptor binding study of true type and wild type AAV2. Authors: Antonette Bennett / Joshua Hull / Nelly Jolinon / Julie Tordo / Katie Moss / Enswert Binns / Mario Mietzsch / Cathleen Hagemann / R Michael Linden / Andrea Serio / Paul Chipman / Duncan ...Authors: Antonette Bennett / Joshua Hull / Nelly Jolinon / Julie Tordo / Katie Moss / Enswert Binns / Mario Mietzsch / Cathleen Hagemann / R Michael Linden / Andrea Serio / Paul Chipman / Duncan Sousa / Felix Broecker / Peter Seeberger / Els Henckaerts / Robert McKenna / Mavis Agbandje-McKenna / Abstract: Adeno-associated viruses (AAV) are utilized as gene transfer vectors in the treatment of monogenic disorders. A variant, rationally engineered based on natural AAV2 isolates, designated AAV-True Type ...Adeno-associated viruses (AAV) are utilized as gene transfer vectors in the treatment of monogenic disorders. A variant, rationally engineered based on natural AAV2 isolates, designated AAV-True Type (AAV-TT), is highly neurotropic compared to wild type AAV2 in vivo, and vectors based on it, are currently being evaluated for central nervous system applications. AAV-TT differs from AAV2 by 14 amino acids, including R585S and R588T, two residues previously shown to be essential for heparan sulfate binding of AAV2. The capsid structures of AAV-TT and AAV2 visualized by cryo-electron microscopy at 3.4 and 3.0 Å resolution, respectively, highlighted structural perturbations at specific amino acid differences. Differential scanning fluorimetry (DSF) performed at different pH conditions demonstrated that the melting temperature (T) of AAV2 was consistently ∼5 °C lower than AAV-TT, but both showed maximal stability at pH 5.5, corresponding to the pH in the late endosome, proposed as required for VP1u externalization to facilitate endosomal escape. Reintroduction of arginines at positions 585 and 588 in AAV-TT caused a reduction in T, demonstrating that the lack of basic amino acids at these positions are associated with capsid stability. These results provide structural and thermal annotation of AAV2/AAV-TT residue differences, that account for divergent cell binding, transduction, antigenic reactivity, and transduction of permissive tissues between the two viruses. Specifically, these data indicate that AAV-TT may not utilize a glycan receptor mediated pathway to enter cells and may have lower antigenic properties as compared to AAV2. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7na6.cif.gz | 5.6 MB | Display | PDBx/mmCIF format |
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PDB format | pdb7na6.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 7na6.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/na/7na6 ftp://data.pdbj.org/pub/pdb/validation_reports/na/7na6 | HTTPS FTP |
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-Related structure data
Related structure data | 24266MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 58482.258 Da / Num. of mol.: 60 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Adeno-associated virus / Gene: cap / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q670S0 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Adeno-associated virus / Type: VIRUS / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Value: 4 MDa / Experimental value: NO |
Source (natural) | Organism: Adeno-associated virus |
Source (recombinant) | Organism: Spodoptera frugiperda (fall armyworm) / Cell: SF9 |
Details of virus | Empty: YES / Enveloped: NO / Isolate: OTHER / Type: VIRION |
Virus shell | Diameter: 260 nm / Triangulation number (T number): 1 |
Buffer solution | pH: 7.4 |
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy |
Image recording | Electron dose: 59 e/Å2 / Detector mode: COUNTING / Film or detector model: DIRECT ELECTRON DE-64 (8k x 8k) / Num. of real images: 1444 |
-Processing
Software | Name: PHENIX / Version: 1.10-2155_2155: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 14778 | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9241 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | B value: 25 / Protocol: FLEXIBLE FIT | ||||||||||||||||||||||||
Refine LS restraints |
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