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- EMDB-6470: Single particle reconstruction of AAV-DJ in complex with ARIXTRA -

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Basic information

Entry
Database: EMDB / ID: EMD-6470
TitleSingle particle reconstruction of AAV-DJ in complex with ARIXTRA
Map dataReconstruction of adeno-associated virus variant DJ in complex with Arixtra
Sample
  • Sample: Adeno-associated virus
  • Virus: Adeno-associated virus
  • Ligand: Fondaparinux
Keywordsgene therapy
Biological speciesunidentified (others) / Adeno-associated virus
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsSpear JM / Noble AJ / Xie Q / Sousa DR / Chapman MS / Stagg SM
CitationJournal: J Struct Biol / Year: 2015
Title: The influence of frame alignment with dose compensation on the quality of single particle reconstructions.
Authors: John M Spear / Alex J Noble / Qing Xie / Duncan R Sousa / Michael S Chapman / Scott M Stagg /
Abstract: As direct electron detection devices in cryo-electron microscopy become ubiquitous, the field is now ripe for new developments in image analysis techniques that take advantage of their increased SNR ...As direct electron detection devices in cryo-electron microscopy become ubiquitous, the field is now ripe for new developments in image analysis techniques that take advantage of their increased SNR coupled with their high-throughput frame collection abilities. In approaching atomic resolution of native-like biomolecules, the accurate extraction of structural locations and orientations of side-chains from frames depends not only on the electron dose that a sample receives but also on the ability to accurately estimate the CTF. Here we use a new 2.8Å resolution structure of a recombinant gene therapy virus, AAV-DJ with Arixtra, imaged on an FEI Titan Krios with a DE-20 direct electron detector to probe new metrics including relative side-chain density and ResLog analysis for optimizing the compensation of electron beam damage and to characterize the factors that are limiting the resolution of the reconstruction. The influence of dose compensation on the accuracy of CTF estimation and particle classifiability are also presented. We show that rigorous dose compensation allows for better particle classifiability and greater recovery of structural information from negatively charged, electron-sensitive side-chains, resulting in a more accurate macromolecular model.
History
DepositionSep 17, 2015-
Header (metadata) releaseOct 7, 2015-
Map releaseOct 7, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing 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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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6470.gif

Downloads & links

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Map

FileDownload / File: emd_6470.map.gz / Format: CCP4 / Size: 300.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of adeno-associated virus variant DJ in complex with Arixtra
Voxel sizeX=Y=Z: 1.215 Å
Density
Contour LevelBy AUTHOR: 0.09 / Movie #1: 0.09
Minimum - Maximum-0.30731693 - 0.56448966
Average (Standard dev.)-0.01868093 (±0.02429028)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 524.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2151.2151.215
M x/y/z432432432
origin x/y/z0.0000.0000.000
length x/y/z524.880524.880524.880
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS432432432
D min/max/mean-0.3070.564-0.019

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Supplemental data

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Sample components

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Entire : Adeno-associated virus

EntireName: Adeno-associated virus
Components
  • Sample: Adeno-associated virus
  • Virus: Adeno-associated virus
  • Ligand: Fondaparinux

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Supramolecule #1000: Adeno-associated virus

SupramoleculeName: Adeno-associated virus / type: sample / ID: 1000
Oligomeric state: 60 viral subunits form the icosahedral capsid
Number unique components: 2
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 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: Yes / Sci species serotype: DJ
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Host systemOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: SF9 / Recombinant plasmid: bacmid
Molecular weightExperimental: 3.75 MDa
Virus shellShell ID: 1 / Name: Shell 1 / Diameter: 250 Å / T number (triangulation number): 1

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Macromolecule #1: Fondaparinux

MacromoleculeName: Fondaparinux / type: ligand / ID: 1 / Name.synonym: Arixtra
Details: Arixtra was added in a 10X molar excess to AAV-DJ monomers
Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)
Molecular weightExperimental: 1.727 KDa
Chemical component information


ChemComp, No image

ChemComp-PRD_900028:
Unknown entry

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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

Concentration0.6 mg/mL
BufferpH: 7.4 / Details: 50mM HEPES, 25mM MgCl2, 25mM NaCl
GridDetails: R2/2 200 mesh copper grids that were rendered hydrophilic in 75/25 % Ar/O.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV
Details: 3ul of 0.6 mg/ml AAV-DJ was applied to grid and then hand blotted. After initial hand blotting, 3ul of 5.7mM Arixtra was added, the grid was mechanically blotted, and then vitrified.
Method: blot force = 1, blot time = 3 seconds, total blots = 1.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49383 / 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: liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: Astigmatism was corrected at 155000 times magnification
DateJan 6, 2015
Image recordingCategory: CCD / Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Number real images: 1051 / Average electron dose: 66 e/Å2 / Details: every image is the sum of 45 frames / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: by image
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: OTHER / Software - Name: EMAN, Frealign / Number images used: 120166
DetailsParticles were picked using an automatic selection program. Particles over carbon were manually deselected.
FSC plot (resolution estimation)

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