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

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Basic information

Entry
Database: PDB / ID: 7kfr
TitleAdeno-Associated Virus (AAV-DJ) - cryo-EM structure at 1.56 Angstrom Resolution
ComponentsCapsid protein VP1
KeywordsVIRUS / Gene therapy vector / VIRUS LIKE PARTICLE
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
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.56 Å
AuthorsXie, Q. / Yoshioka, C.K. / Chapman, M.S.
Funding support United States, 1items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 16, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_oper_list
Item: _citation.country / _database_2.pdbx_DOI ..._citation.country / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Capsid protein VP1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)58,9514
Polymers58,8781
Non-polymers733
Water4,774265
1
A: Capsid protein VP1
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)3,537,043240
Polymers3,532,66960
Non-polymers4,375180
Water1,08160
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: Capsid protein VP1
hetero molecules
x 5


  • icosahedral pentamer
  • 295 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)294,75420
Polymers294,3895
Non-polymers36515
Water905
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Capsid protein VP1
hetero molecules
x 6


  • icosahedral 23 hexamer
  • 354 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)353,70424
Polymers353,2676
Non-polymers43718
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein Capsid protein VP1


Mass: 58877.809 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus / Gene: cap / Variant: DJ / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6JC41
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 265 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Adeno-associated virus / Type: VIRUS / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 3.746 MDa / Experimental value: NO
Source (natural)Organism: Adeno-associated virus
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Details of virusEmpty: YES / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Homo sapiens
Virus shellDiameter: 250 nm / Triangulation number (T number): 1
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
225 mMMagnesium chlorideMgCl21
325 mMSodium chlorideNaCl1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse
Specimen supportDetails: PELCO easiGlow Glow Discharge Cleaning System Current: 15mA
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K
Details: Two 3uL aliquots applied to grid (manual blotting between), prior to automated 3 second blot before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Coma-free alignment and objective astigmatism where corrected using Sherpa (Thermo Fisher, Inc.).
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 155000 X / Calibrated magnification: 154400 X / Nominal defocus max: -2600 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 93 K / Temperature (min): 93 K
Image recordingAverage exposure time: 15.8 sec. / Electron dose: 30 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2241
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.
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
1DoG Pickerparticle selection
2EPUimage acquisition
4GctfCTF correctionImplemented through wrapper within Relion 3.0
7Coot0.8.9.2model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13RSRef0.5.8model refinement
14CNS1.3model refinementwith embedded RSRef 0.5.6
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles 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.
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 1.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48209
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.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Least-squares residual
Details: Stand-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 ...Details: Stand-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.
Atomic model buildingPDB-ID: 5UF6

5uf6


Accession code: 5UF6 / Source name: PDB / Type: experimental model

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