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- EMDB-5424: Structure of adeno-associated virus-2 in complex with neutralizin... -

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Basic information

Entry
Database: EMDB / ID: EMD-5424
TitleStructure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20
Map dataReconstruction of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20
Sample
  • Sample: Adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20
  • Virus: Adeno-associated virus - 2
KeywordsAdeno-associated virus / Antibody / A20 / Epitope / Fab / Gene therapy / Monoclonal
Function / homology
Function and homology information


symbiont entry into host cell via permeabilization of host membrane / host cell nucleolus / T=1 icosahedral viral capsid / clathrin-dependent endocytosis of virus by host cell / virion attachment to host cell / structural molecule activity
Similarity search - Function
Phospholipase A2-like domain / Phospholipase A2-like domain / Parvovirus coat protein VP2 / Parvovirus coat protein VP1/VP2 / Parvovirus coat protein VP1/VP2 / Capsid/spike protein, ssDNA virus
Similarity search - Domain/homology
Biological speciesAdeno-associated virus - 2
Methodsingle particle reconstruction / cryo EM / Resolution: 8.5 Å
AuthorsMcCraw DM / O'Donnell JK / Taylor KA / Stagg SM / Chapman MS
CitationJournal: Virology
Title: Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20.
Authors: Dustin M McCraw / Jason K O'Donnell / Kenneth A Taylor / Scott M Stagg / Michael S Chapman /
Abstract: The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5Å resolution is determined for ...The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5Å resolution is determined for a complex of AAV-2 with the Fab' fragment of monoclonal antibody (MAb) A20, the most extensively characterized AAV MAb. The binding footprint is determined through fitting the cryo-EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. The footprint extends from the previously implicated plateau to the side of the spike, and into the conserved canyon, covering a larger area than anticipated. Comparison with structures of binding and non-binding serotypes indicates that recognition depends on a combination of subtle serotype-specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune-resistant vectors that can still bind to target cells.
History
DepositionMay 22, 2012-
Header (metadata) releaseMay 29, 2012-
Map releaseMay 29, 2012-
UpdateJun 12, 2012-
Current statusJun 12, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j1s
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j1s
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5424_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5424.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.45 Å/pix.
x 240 pix.
= 588. Å		2.45 Å/pix.
x 240 pix.
= 588. Å		2.45 Å/pix.
x 240 pix.
= 588. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.45 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.34 / Movie #1: 1.5
Minimum - Maximum-8.442361829999999 - 9.859992979999999
Average (Standard dev.)0.01103545 (±0.99768275)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-120
Dimensions240240240
Spacing240240240
CellA=B=C: 588.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.452.452.45
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z588.000588.000588.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-1500
NX/NY/NZ301301151
MAP C/R/S123
start NC/NR/NS-120-120-120
NC/NR/NS240240240
D min/max/mean-8.4429.8600.011

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Supplemental data

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Sample components

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Entire : Adeno-associated virus-2 in complex with neutralizing monoclonal ...

EntireName: Adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20
Components
  • Sample: Adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20
  • Virus: Adeno-associated virus - 2

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Supramolecule #1000: Adeno-associated virus-2 in complex with neutralizing monoclonal ...

SupramoleculeName: Adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20
type: sample / ID: 1000
Oligomeric state: 60 A20 Fab's bind to one adeno-associated virus (one adeno-associated virus consists of 60 viral proteins)
Number unique components: 2
Molecular weightTheoretical: 6.9 MDa

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Supramolecule #1: Adeno-associated virus - 2

SupramoleculeName: Adeno-associated virus - 2 / type: virus / ID: 1 / Name.synonym: AAV-2 / NCBI-ID: 10804 / Sci species name: Adeno-associated virus - 2 / Database: NCBI / Virus type: VIRION / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No / Syn species name: AAV-2
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Molecular weightTheoretical: 3.9 MDa
Virus shellShell ID: 1 / Diameter: 280 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

Concentration0.14 mg/mL
BufferpH: 7.2
Details: 100 mM HEPES, 50 mM magnesium chloride, and 5% glycerol
GridDetails: 400 mesh carbon grid with holey carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 2.0 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
Legacy - Electron beam tilt params: 0
DateFeb 23, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Number real images: 1503 / Average electron dose: 15 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 39775 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -4.0 µm / Nominal defocus min: -0.5 µm / Nominal magnification: 37000
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Whole image
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Appion, ACE, EMAN / Number images used: 11898
Final two d classificationNumber classes: 304

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Atomic model buiding 1

Initial modelPDB ID:

1lp3


Chain - Chain ID: A
SoftwareName: RSRef
DetailsProtocol: Fixed. Anti-bumping restraints from CNS included 1LP3, Constrained icosahedral symmetry
RefinementSpace: REAL / Overall B value: 0
Target criteria: Least-squares difference between experimental & model coulombic potential
Output model

PDB-3j1s:
Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20

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