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- EMDB-5415: Structure of AAV-DJ, a Retargeted Gene Therapy Vector: Cryo-Elect... -

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Basic information

Entry
Database: EMDB / ID: EMD-5415
TitleStructure of AAV-DJ, a Retargeted Gene Therapy Vector: Cryo-Electron Microscopy at 4.5A resolution
Map dataReconstruction of AAV-DJ, a re-targeted gene therapy vector
Sample
  • Sample: AAV-DJ
  • Virus: Adeno-associated virus
KeywordsGene therapy / adeno-associated virus / vector / cryo-electron microscopy / directed evolution / AAV
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
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsLerch TF / O'Donnell JK / Meyer NL / Xie Q Taylor KA / Stagg SC / Chapman MS
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, 2012-
Header (metadata) releaseAug 22, 2012-
Map releaseAug 22, 2012-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j1q
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j1q
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5415_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5415.map.gz / Format: CCP4 / Size: 173.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of AAV-DJ, a re-targeted gene therapy vector
Voxel sizeX=Y=Z: 1.3217 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.1243183 - 0.17182954
Average (Standard dev.)-0.02058947 (±0.01350072)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-180-180-180
Dimensions360360360
Spacing360360360
CellA=B=C: 475.81198 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.32171.32171.3217
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z475.812475.812475.812
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-180-180-180
NC/NR/NS360360360
D min/max/mean-0.1240.172-0.021

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Supplemental data

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Sample components

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Entire : AAV-DJ

EntireName: AAV-DJ
Components
  • Sample: AAV-DJ
  • Virus: Adeno-associated virus

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Supramolecule #1000: AAV-DJ

SupramoleculeName: AAV-DJ / type: sample / ID: 1000
Oligomeric state: 60 viral subunits form the icosahedral capsid
Number unique components: 1
Molecular weightTheoretical: 3.75 MDa

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Supramolecule #1: Adeno-associated virus

SupramoleculeName: Adeno-associated virus / type: virus / ID: 1 / NCBI-ID: 272636 / Sci species name: Adeno-associated virus / Database: NCBI / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Molecular weightTheoretical: 3.75 MDa
Virus shellShell ID: 1 / Name: VP3 / Diameter: 250 Å / T number (triangulation number): 1

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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

Concentration1.5 mg/mL
BufferpH: 7.5 / Details: 125mM NaCl, 10mM Tris, 1mM MgCl2, pH 7.5
GridDetails: C-flat grid CF-2/1-2C (2um hole, 1 um spacing, 200 mesh copper), glow discharged
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK I

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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
DateFeb 25, 2011
Image recordingNumber real images: 4773
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTF was estimated using Appion software
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN / Number images used: 27312
DetailsImages were collected automatically using Leginon software. Particles were manually selected for single particle reconstruction.

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Atomic model buiding 1

Initial modelPDB ID:

1lp3


Chain - Chain ID: A
SoftwareName: RSRef, CNS
DetailsProtocol: Refinement of reconstruction magnification relative to homolog crystal structure, manual rebuilding, simulated annealing torsion angle dynamics, isotropic B-factors. Iterative refinement in RSRef and model building in Coot
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 30 / Target criteria: Correlation coefficient
Output model

PDB-3j1q:
Structure of AAV-DJ, a Retargeted Gene Therapy Vector: Cryo-Electron Microscopy at 4.5A resolution

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