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- PDB-3j1q: Structure of AAV-DJ, a Retargeted Gene Therapy Vector: Cryo-Elect... -

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Basic information

Entry
Database: PDB / ID: 3j1q
TitleStructure of AAV-DJ, a Retargeted Gene Therapy Vector: Cryo-Electron Microscopy at 4.5A resolution
ComponentsAdeno-associated virus DJ
KeywordsVIRUS / Gene therapy
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 VP1
Function and homology information
Biological speciesAdeno-associated virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsLerch, T.F. / O'Donnell, J.K. / Meyer, N.L. / Xie, Q. / Taylor, K.A. / Stagg, S.M. / Chapman, M.S.
CitationJournal: Structure / Year: 2012
Title: Structure of AAV-DJ, a retargeted gene therapy vector: cryo-electron microscopy at 4.5 Å resolution.
Authors: Thomas F Lerch / Jason K O'Donnell / Nancy L Meyer / Qing Xie / Kenneth A Taylor / Scott M Stagg / Michael S Chapman /
Abstract: AAV-DJ, a leading candidate vector for liver gene therapy, was created through random homologous recombination followed by directed evolution, selecting for in vivo liver tropism and resistance to ...AAV-DJ, a leading candidate vector for liver gene therapy, was created through random homologous recombination followed by directed evolution, selecting for in vivo liver tropism and resistance to in vitro immune neutralization. Here, the 4.5 Å resolution cryo-EM structure is determined for the engineered AAV vector, revealing structural features that illuminate its phenotype. The heparan sulfate receptor-binding site is little changed from AAV-2, and heparin-binding affinity is similar. A loop that is antigenic in other serotypes has a unique conformation in AAV-DJ that would conflict with the binding of an AAV-2 neutralizing monoclonal antibody. This is consistent with increased resistance to neutralization by human polyclonal sera, raising the possibility that changed tropism may be a secondary effect of altered immune interactions. The reconstruction exemplifies analysis of fine structural changes and the potential of cryo-EM, in favorable cases, to characterize mutant or ligand-bound complexes.
History
DepositionApr 30, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 22, 2012Provider: repository / Type: Initial release
Revision 1.1Oct 24, 2012Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.3Feb 21, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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  • 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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Structure viewerMolecule:
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Assembly

Deposited unit
A: Adeno-associated virus DJ


Theoretical massNumber of molelcules
Total (without water)82,0081
Polymers82,0081
Non-polymers00
Water0
1
A: Adeno-associated virus DJ
x 60


Theoretical massNumber of molelcules
Total (without water)4,920,50460
Polymers4,920,50460
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: Adeno-associated virus DJ
x 5


  • icosahedral pentamer
  • 410 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)410,0425
Polymers410,0425
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Adeno-associated virus DJ
x 6


  • icosahedral 23 hexamer
  • 492 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)492,0506
Polymers492,0506
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 Adeno-associated virus DJ / AAV-DJ


Mass: 82008.406 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus / Strain: hybrid of serotypes 2, 8 and 9 / Gene: Capsid / Plasmid: pFBDDJm11 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): SF9 / References: UniProt: Q6JC41*PLUS

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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 DJ / Type: COMPLEX
Details: 60 subunits associate to form a T=1 icosahedral capsid
Details of virusHost category: VERTEBRATES / Isolate: SEROTYPE / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Homo sapiens
Buffer solutionName: 125 mM sodium chloride, 10 mM Tris, 1 mM magnesium chloride
pH: 7.5
Details: 125 mM sodium chloride, 10 mM Tris, 1 mM magnesium chloride
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Sample prepared on C-flat 2um hole, 1 um spacing, 200 mesh copper grids
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Feb 25, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderSpecimen holder model: OTHER
Image scansNum. digital images: 4773
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategoryDetails
1CNSmodel fitting
2RSRefmodel fittingRSRef embedded in CNS
3FREALIGN3D reconstruction
CTF correctionDetails: CTF was estimated using Appion software
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: single particle, icosahedralSingle particle analysis
Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27312 / Nominal pixel size: 1.3217 Å / Actual pixel size: 1.3217 Å
Magnification calibration: Magnification was calibrated against the crystal structure of a known homolog, AAV2 (1LP3)
Details: Frealign was used for particle refinement in the reconstruction
Symmetry type: POINT
Atomic model buildingB value: 30 / Protocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient
Details: REFINEMENT PROTOCOL--simulated annealing torsion angle dynamics, isotropic B-factor refinement DETAILS--Iterative refinement in RSRef and model building in Coot
Atomic model buildingPDB-ID: 1LP3

1lp3


Pdb chain-ID: A / Accession code: 1LP3 / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms4136 0 0 0 4136

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