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- PDB-7kp3: Adeno-associated virus serotype 5 at 2.1 Angstroms resolution, AAV5 -

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Basic information

Entry
Database: PDB / ID: 7kp3
TitleAdeno-associated virus serotype 5 at 2.1 Angstroms resolution, AAV5
ComponentsCapsid proteinCapsid
KeywordsVIRUS / AAV5 / AAV / AAV-5 / Adeno Associated Virus / VIRUS LIKE PARTICLE / parvovirus / gene therapy
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
Function and homology information
Biological speciesAdeno-associated virus - 5
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsSilveria, M. / 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: The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors.
Authors: Mark A Silveria / Edward E Large / Grant M Zane / Tommi A White / Michael S Chapman /
Abstract: Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology ...Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.
History
DepositionNov 10, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 16, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2023Group: Database references / Derived calculations / Refinement description
Category: citation / database_2 ...citation / database_2 / pdbx_initial_refinement_model / pdbx_struct_oper_list / refine
Item: _citation.country / _database_2.pdbx_DOI ..._citation.country / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type / _refine.ls_d_res_high
Revision 1.2May 29, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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

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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


Theoretical massNumber of molelcules
Total (without water)80,3661
Polymers80,3661
Non-polymers00
Water0
1
A: Capsid protein
x 60


Theoretical massNumber of molelcules
Total (without water)4,821,97360
Polymers4,821,97360
Non-polymers00
Water0
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
x 5


  • icosahedral pentamer
  • 402 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)401,8315
Polymers401,8315
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Capsid protein
x 6


  • icosahedral 23 hexamer
  • 482 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)482,1976
Polymers482,1976
Non-polymers00
Water0
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 / Capsid


Mass: 80366.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus - 5 / Gene: cap, VP1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9YIJ1

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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
Details: Expressed using SF9 cells with a pfastbac LIC vector. Purified with cesium chloride ultracentrifugation.
Entity ID: all / 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 chlorideNaClSodium chloride1
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K
Details: Two 2uL 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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: -2700 nm / Nominal defocus min: -700 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 recordingElectron dose: 31.7 e/Å2 / Detector mode: INTEGRATING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9490 / Details: Pixel size was 0.5295 angstrom.
Image scansWidth: 11520 / Height: 8184

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.1 betaparticle selection
2EPUimage acquisition
4CTFFIND4CTF correctionImplemented through wrapper within Relion 3.1 beta
7Coot0.8.9.2model fitting
9RELION3.1 betainitial Euler assignment
10RELION3.1 betafinal Euler assignment
11RELION3.1 betaclassification
12RELION3.1 Beta3D reconstruction
13RSRef0.5.8model refinement
14CNS1.3model refinementwith embedded RSRef 0.5.6
CTF correctionDetails: CTF correction was performed on a per particle basis.
Type: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 672106
Details: LoG Picker was used for initial automated particle selection. Templates were then generated by 2D classification, followed by particle template selection in Relion 3.1 beta.
3D reconstructionResolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 373426 / 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: 3NTT

3ntt


Accession code: 3NTT / Source name: PDB / Type: experimental model
RefinementHighest resolution: 2.1 Å

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