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- EMDB-22854: Adeno-Associated Virus (AAV-DJ) - cryo-EM structure at 1.56 Angst... -

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Basic information

Entry
Database: EMDB / ID: EMD-22854
TitleAdeno-Associated Virus (AAV-DJ) - cryo-EM structure at 1.56 Angstrom Resolution
Map dataFull particle Relion map, sharpened using volume-whitening routine of cisTEM. Cropped version also provided for ease of comparison with coordinates.
Sample
  • Virus: Adeno-associated virus
    • Protein or peptide: Capsid protein VP1
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsGene therapy vector / VIRUS LIKE PARTICLE / VIRUS
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: 1.56 Å
AuthorsXie Q / Yoshioka CK
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122564 United States
CitationJournal: Viruses / Year: 2020
Title: Adeno-Associated Virus (AAV-DJ)-Cryo-EM Structure at 1.56 Å Resolution.
Authors: Qing Xie / Craig K Yoshioka / Michael S Chapman /
Abstract: Adeno-associated virus is the leading viral vector for gene therapy. AAV-DJ is a recombinant variant developed for tropism to the liver. The AAV-DJ structure has been determined to 1.56 Å resolution ...Adeno-associated virus is the leading viral vector for gene therapy. AAV-DJ is a recombinant variant developed for tropism to the liver. The AAV-DJ structure has been determined to 1.56 Å resolution through cryo-electron microscopy (cryo-EM). Only apoferritin is reported in preprints at 1.6 Å or higher resolution, and AAV-DJ nearly matches the highest resolutions ever attained through X-ray diffraction of virus crystals. However, cryo-EM has the advantage that most of the hydrogens are clear, improving the accuracy of atomic refinement, and removing ambiguity in hydrogen bond identification. Outside of secondary structures where hydrogen bonding was predictable a priori, the networks of hydrogen bonds coming from direct observation of hydrogens and acceptor atoms are quite different from those inferred even at 2.8 Å resolution. The implications for understanding viral assembly mean that cryo-EM will likely become the favored approach for high resolution structural virology.
History
DepositionOct 14, 2020-
Header (metadata) releaseDec 16, 2020-
Map releaseDec 16, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kfr
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7kfr
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22854.png

Downloads & links

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Map

FileDownload / File: emd_22854.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull particle Relion map, sharpened using volume-whitening routine of cisTEM. Cropped version also provided for ease of comparison with coordinates.
Voxel sizeX=Y=Z: 0.5105 Å
Density
Contour LevelBy EMDB: 0.064 / Movie #1: 0.04
Minimum - Maximum-0.177237 - 0.372257
Average (Standard dev.)-0.000013357471 (±0.017671466)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-300-300-300
Dimensions600600600
Spacing600600600
CellA=B=C: 306.30002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.51050.51050.5105
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z306.300306.300306.300
α/β/γ90.00090.00090.000
start NX/NY/NZ-300-300-300
NX/NY/NZ600600600
MAP C/R/S213
start NC/NR/NS-300-300-300
NC/NR/NS600600600
D min/max/mean-0.1770.372-0.000

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Supplemental data

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Mask #1

Fileemd_22854_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Full particle, unsharpened Relion map, low pass filtered...

Fileemd_22854_additional_1.map
AnnotationFull particle, unsharpened Relion map, low pass filtered beyond FSC of 1.56 A. See also cropped version.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened Relion map, cropped ~20 Angstrom outside subunit...

Fileemd_22854_additional_2.map
AnnotationUnsharpened Relion map, cropped ~20 Angstrom outside subunit coordinates deposited, for ease of visualization.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Relion map, sharpened using volume-whitening routine of cisTEM,...

Fileemd_22854_additional_3.map
AnnotationRelion map, sharpened using volume-whitening routine of cisTEM, and cropped ~20 Angstrom outside deposited subunit coordinates for ease of comparison.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Relion half map 1.

Fileemd_22854_half_map_1.map
AnnotationRelion half map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Relion half map 2.

Fileemd_22854_half_map_2.map
AnnotationRelion half map 2.
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
    • Protein or peptide: Capsid protein VP1
  • Ligand: MAGNESIUM ION
  • Ligand: water

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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 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.746 MDa
Virus shellShell ID: 1 / Diameter: 250.0 Å / T number (triangulation number): 1

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Macromolecule #1: Capsid protein VP1

MacromoleculeName: Capsid protein VP1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Adeno-associated virus
Molecular weightTheoretical: 58.877809 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GADGVGNSSG NWHCDSTWMG DRVITTSTRT WALPTYNNHL YKQISNSTSG GSSNDNAYFG YSTPWGYFDF NRFHCHFSPR DWQRLINNN WGFRPKRLSF KLFNIQVKEV TQNEGTKTIA NNLTSTIQVF TDSEYQLPYV LGSAHQGCLP PFPADVFMIP Q YGYLTLNN ...String:
GADGVGNSSG NWHCDSTWMG DRVITTSTRT WALPTYNNHL YKQISNSTSG GSSNDNAYFG YSTPWGYFDF NRFHCHFSPR DWQRLINNN WGFRPKRLSF KLFNIQVKEV TQNEGTKTIA NNLTSTIQVF TDSEYQLPYV LGSAHQGCLP PFPADVFMIP Q YGYLTLNN GSQAVGRSSF YCLEYFPSQM LRTGNNFQFT YTFEDVPFHS SYAHSQSLDR LMNPLIDQYL YYLSRTQTTG GT TNTQTLG FSQGGPNTMA NQAKNWLPGP CYRQQRVSKT SADNNNSEYS WTGATKYHLN GRDSLVNPGP AMASHKDDEE KFF PQSGVL IFGKQGSEKT NVDIEKVMIT DEEEIRTTNP VATEQYGSVS TNLQRGNRQA ATADVNTQGV LPGMVWQDRD VYLQ GPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPADPPT TFNQSKLNSF ITQYSTGQVS VEIEWELQKE NSKRW NPEI QYTSNYYKST SVDFAVNTEG VYSEPRPIGT RYLTRNL

UniProtKB: Capsid protein VP1

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 265 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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
Component:
ConcentrationNameFormula
50.0 mMHEPES
25.0 mMMagnesium chlorideMgCl2
25.0 mMSodium chlorideNaClSodium chloride
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.03 kPa
Details: PELCO easiGlow Glow Discharge Cleaning System Current: 15mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
Details: Two 3uL aliquots applied to grid (manual blotting between), prior to automated 3 second blot before plunging..
DetailsMonodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 154400 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -2.6 µm / Nominal defocus min: -0.8 µm / Nominal magnification: 155000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 93.0 K / Max: 93.0 K
DetailsComa-free alignment and objective astigmatism where corrected using Sherpa (Thermo Fisher, Inc.).
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 2241 / Average exposure time: 15.8 sec. / Average electron dose: 30.0 e/Å2
Details: Data were collected with pixel size of 0.514 angstrom on a FEI Titan Krios (Thermo Fisher, Inc.) at 300 kV, using a Falcon 3 camera (Thermo Fisher) with a total dose of approx. 30 e-/A2 ...Details: Data were collected with pixel size of 0.514 angstrom on a FEI Titan Krios (Thermo Fisher, Inc.) at 300 kV, using a Falcon 3 camera (Thermo Fisher) with a total dose of approx. 30 e-/A2 fractionated across 200 frames. Camera dose rate was approx. 0.5 e-/pixel/s. Defocus was random in the nominal range of -0.8 to -2.6 um. Images were acquired in EPU (Thermo Fisher, Inc.) without using image shift.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 75316
Details: DoG (Difference of Gaussian) Picker was used for initial automated particle selection. Templates were then generated by 2D classification, followed by particle template selection in Relion 3.0.
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Details: Multiple rounds of 2D classification and 3D classification with C1 symmetry were used to remove outliers, resulting in 48,209 particles after deduplication.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 1.56 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0)
Details: These 48209 particles refined to approx. 2.2 A with I1 symmetry, and subsequent refinements of beam tilt and per-particle CTF brought the resolution to 1.8 A. Further particle-polishing and ...Details: These 48209 particles refined to approx. 2.2 A with I1 symmetry, and subsequent refinements of beam tilt and per-particle CTF brought the resolution to 1.8 A. Further particle-polishing and subsequent re-refinement of CTF brought the resolution to 1.70 A and a final reconstruction using Ewald sphere correction ended at 1.56 A. The map used for modeling was sharpened using the volume whitening routine in cisTEM.
Number images used: 48209

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Atomic model buiding 1

Initial modelPDB ID:

5uf6


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsStand-alone RSRef was used for refinement of magnification, resolution, envelope correction and atomic B-factors. This was alternated with RSRef-embedded CNS was used for molecular dynamics optimization (1st round) and stereochemically-restrained all-atom least-squares optimization.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Least-squares residual
Output model

PDB-7kfr:
Adeno-Associated Virus (AAV-DJ) - cryo-EM structure at 1.56 Angstrom Resolution

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