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- EMDB-6470: Single particle reconstruction of AAV-DJ in complex with ARIXTRA -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-6470 | |||||||||
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Title | Single particle reconstruction of AAV-DJ in complex with ARIXTRA | |||||||||
![]() | Reconstruction of adeno-associated virus variant DJ in complex with Arixtra | |||||||||
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![]() | gene therapy | |||||||||
Biological species | unidentified (others) / ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.8 Å | |||||||||
![]() | Spear JM / Noble AJ / Xie Q / Sousa DR / Chapman MS / Stagg SM | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 258.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 11.2 KB 11.2 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 17.7 KB | Display | ![]() |
Images | ![]() ![]() | 90.7 KB 4.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | Reconstruction of adeno-associated virus variant DJ in complex with Arixtra | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.215 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Adeno-associated virus
Entire | Name: ![]() ![]() |
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Components |
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-Supramolecule #1000: Adeno-associated virus
Supramolecule | Name: Adeno-associated virus / type: sample / ID: 1000 Oligomeric state: 60 viral subunits form the icosahedral capsid Number unique components: 2 |
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Molecular weight | Theoretical: 3.75 MDa |
-Supramolecule #1: Adeno-associated virus
Supramolecule | Name: 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 |
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Host (natural) | Organism: ![]() |
Host system | Organism: ![]() ![]() |
Molecular weight | Experimental: 3.75 MDa |
Virus shell | Shell ID: 1 / Name: Shell 1 / Diameter: 250 Å / T number (triangulation number): 1 |
-Macromolecule #1: Fondaparinux
Macromolecule | Name: 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 |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Experimental: 1.727 KDa |
Chemical component information | ![]()
ChemComp-PRD_900028: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.6 mg/mL |
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Buffer | pH: 7.4 / Details: 50mM HEPES, 25mM MgCl2, 25mM NaCl |
Grid | Details: R2/2 200 mesh copper grids that were rendered hydrophilic in 75/25 % Ar/O. |
Vitrification | Cryogen 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
Microscope | FEI TITAN KRIOS |
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Temperature | Average: 93 K |
Alignment procedure | Legacy - Astigmatism: Astigmatism was corrected at 155000 times magnification |
Date | Jan 6, 2015 |
Image recording | Category: 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 49383 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 29000 |
Sample stage | Specimen holder: liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |