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- EMDB-8574: Single particle reconstruction of chimeric adeno-associated virus... -

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Basic information

Entry
Database: EMDB / ID: EMD-8574
TitleSingle particle reconstruction of chimeric adeno-associated virus-DJ with a Heparanoid Pentasaccharide
Map datachimeric adeno-associated virus-DJ with a Heparanoid Pentasaccharide
Sample
  • Virus: Adeno-associated virus
Biological speciesAdeno-associated virus
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsXie Q / Noble AJ / Sousa DR / Meyer NL / Davulcu O / Zhang FM / Linhardt RJ / Stagg SM / Chapman MS
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM66875 United States
CitationJournal: Mol Ther Methods Clin Dev / Year: 2017
Title: The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparinoid Pentasaccharide.
Authors: Qing Xie / John M Spear / Alex J Noble / Duncan R Sousa / Nancy L Meyer / Omar Davulcu / Fuming Zhang / Robert J Linhardt / Scott M Stagg / Michael S Chapman /
Abstract: Atomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 Å resolution. The gene therapy vector, AAV-DJ, is ...Atomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 Å resolution. The gene therapy vector, AAV-DJ, is a hybrid of natural serotypes that was previously derived by directed evolution, selecting for hepatocyte entry and resistance to neutralization by human serum. The structure of AAV-DJ differs from that of parental serotypes in two regions where neutralizing antibodies bind, so immune escape appears to have been the primary driver of AAV-DJ's directed evolution. Fondaparinux is an analog of cell surface heparan sulfate to which several AAVs bind during entry. Fondaparinux interacts with viral arginines at a known heparin binding site, without the large conformational changes whose presence was controversial in low-resolution imaging of AAV2-heparin complexes. The glycan density suggests multi-modal binding that could accommodate sequence variation and multivalent binding along a glycan polymer, consistent with a role in attachment, prior to more specific interactions with a receptor protein mediating entry.
History
DepositionJan 25, 2017-
Header (metadata) releaseFeb 22, 2017-
Map releaseMay 24, 2017-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5uf6
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5uf6
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8574.png

Downloads & links

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Map

FileDownload / File: emd_8574.map.gz / Format: CCP4 / Size: 76.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationchimeric adeno-associated virus-DJ with a Heparanoid Pentasaccharide
Voxel sizeX=Y=Z: 1.2159 Å
Density
Contour LevelBy AUTHOR: 0.09 / Movie #1: 0.09
Minimum - Maximum-0.30731693 - 0.56448966
Average (Standard dev.)-0.010375952 (±0.047690388)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-136-136-136
Dimensions272272272
Spacing272272272
CellA=B=C: 330.7248 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21590073529411.21590073529411.2159007352941
M x/y/z272272272
origin x/y/z0.0000.0000.000
length x/y/z330.725330.725330.725
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS-136-136-136
NC/NR/NS272272272
D min/max/mean-0.3070.564-0.010

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Supplemental data

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Additional map: Adeno-Associated Virus-DJ Bound by a Heparanoid Pentasaccharide, native...

Fileemd_8574_additional_1.map
AnnotationAdeno-Associated Virus-DJ Bound by a Heparanoid Pentasaccharide, native map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Adeno-Associated Virus-DJ Bound by a Heparanoid Pentasaccharide, difference...

Fileemd_8574_additional_2.map
AnnotationAdeno-Associated Virus-DJ Bound by a Heparanoid Pentasaccharide, difference map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Adeno-associated virus

EntireName: Adeno-associated virus
Components
  • Virus: Adeno-associated virus

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Supramolecule #1: Adeno-associated virus

SupramoleculeName: Adeno-associated virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1 / NCBI-ID: 272636 / Sci species name: Adeno-associated virus / Sci species strain: hybrid of serotypes 2, 8, and 9 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: Yes
Host systemOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant strain: SF9 / Recombinant plasmid: pFBDDJM11
Molecular weightTheoretical: 3.75 MDa

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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

Concentration0.60 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
25.0 mMMgCl2magnesium chloride
25.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: blot force = 1, blot time = 3 seconds, total blots = 1.
Details60 viral subunits form the icosahedral capsid

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Digitization - Frames/image: 1-45 / Number grids imaged: 1 / Number real images: 1051 / Average exposure time: 1.4 sec. / Average electron dose: 66.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 120166
CTF correctionSoftware: (Name: ACE, CTFFIND)
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN / Number images used: 107454

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