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- PDB-5uf6: The 2.8 A Electron Microscopy Structure of Adeno-Associated Virus... -

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Basic information

Entry
Database: PDB / ID: 5uf6
TitleThe 2.8 A Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparanoid Pentasaccharide
Componentscapsid protein VP1
KeywordsVIRUS LIKE PARTICLE / glycan / receptor / attachment
Function / homologyEmpty Capsid Viral Protein 2 / Parvovirus coat protein VP1/VP2 / Beta Complex / Mainly Beta / fondaparinux
Function and homology information
Biological speciesAdeno-associated virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsXie, Q. / Spear, J.M. / Noble, A.J. / Sousa, D.R. / Meyer, N.L. / Davulcu, O. / Zhang, F. / Linhardt, R.J. / Stagg, S.M. / Chapman, M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM66875 United States
CitationJournal: Mol Ther Methods Clin Dev / Year: 2017
Title: The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparinoid Pentasaccharide.
Authors: Qing Xie / John M Spear / Alex J Noble / Duncan R Sousa / Nancy L Meyer / Omar Davulcu / Fuming Zhang / Robert J Linhardt / Scott M Stagg / Michael S Chapman /
Abstract: Atomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 Å resolution. The gene therapy vector, AAV-DJ, is ...Atomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 Å resolution. The gene therapy vector, AAV-DJ, is a hybrid of natural serotypes that was previously derived by directed evolution, selecting for hepatocyte entry and resistance to neutralization by human serum. The structure of AAV-DJ differs from that of parental serotypes in two regions where neutralizing antibodies bind, so immune escape appears to have been the primary driver of AAV-DJ's directed evolution. Fondaparinux is an analog of cell surface heparan sulfate to which several AAVs bind during entry. Fondaparinux interacts with viral arginines at a known heparin binding site, without the large conformational changes whose presence was controversial in low-resolution imaging of AAV2-heparin complexes. The glycan density suggests multi-modal binding that could accommodate sequence variation and multivalent binding along a glycan polymer, consistent with a role in attachment, prior to more specific interactions with a receptor protein mediating entry.
History
DepositionJan 3, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 24, 2017Provider: repository / Type: Initial release
Revision 1.1Jul 12, 2017Group: Structure summary / Item: _chem_comp.pdbx_synonyms
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.3Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / entity_name_com / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_molecule_features / pdbx_nonpoly_scheme / struct_conn / struct_site / struct_site_gen
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.type_symbol / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.pdbx_synonyms / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.src_method / _entity.type
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Mar 6, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / citation / database_2 / pdbx_struct_oper_list
Item: _chem_comp.pdbx_synonyms / _citation.country ..._chem_comp.pdbx_synonyms / _citation.country / _citation.journal_id_ISSN / _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

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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)60,0862
Polymers58,5781
Non-polymers1,5081
Water97354
1
A: capsid protein VP1
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)3,605,148120
Polymers3,514,65260
Non-polymers90,49660
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
  • 300 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)300,42910
Polymers292,8885
Non-polymers7,5415
Water905
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: capsid protein VP1
hetero molecules
x 6


  • icosahedral 23 hexamer
  • 361 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)360,51512
Polymers351,4656
Non-polymers9,0506
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: 58577.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus / Strain: hybrid of serotypes 2, 8, and 9 / Gene: CAPSID / Plasmid: pFBDDJM11 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): SF9
#2: Polysaccharide 2-deoxy-6-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranose-(1-4)-beta-D-glucopyranuronic acid-(1-4)-2- ...2-deoxy-6-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranose-(1-4)-beta-D-glucopyranuronic acid-(1-4)-2-deoxy-3,6-di-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranose-(1-4)-2-O-sulfo-alpha-L-idopyranuronic acid-(1-4)-methyl 2-deoxy-6-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranoside / fondaparinux /


Type: oligosaccharide, Oligosaccharide / Class: Anticoagulant / Mass: 1508.264 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: fondaparinux
DescriptorTypeProgram
WURCS=2.0/5,5,4/[a2122h-1a_1-5_1*OC_2*NSO/3=O/3=O_6*OSO/3=O/3=O][a2121A-1a_1-5_2*OSO/3=O/3=O][a2122h-1a_1-5_2*NSO/3=O/3=O_3*OSO/3=O/3=O_6*OSO/3=O/3=O][a2122A-1b_1-5][a2122h-1a_1-5_2*NSO/3=O/3=O_6*OSO/3=O/3=O]/1-2-3-4-5/a4-b1_b4-c1_c4-d1_d4-e1WURCSPDB2Glycan 1.1.0
[][methyl]{[(1+1)][a-D-GlcpNSO36SO3]{[(4+1)][a-L-IdopA2SO3]{[(4+1)][a-D-GlcpNSO33SO36SO3]{[(4+1)][b-D-GlcpA]{[(4+1)][a-D-GlcpNSO36SO3]{}}}}}}LINUCSPDB-CARE
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 54 / Source method: isolated from a natural source / Formula: H2O

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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: 3.75 MDa / Experimental value: NO
Source (natural)Organism: Adeno-associated virus / Strain: hybrid of serotypes 2, 8, and 9
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: SF9 / Plasmid: pFBDDJM11
Details of virusEmpty: YES / Enveloped: NO / Isolate: SEROTYPE / Type: VIRUS-LIKE PARTICLE
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
225 mMmagnesium chlorideMgCl21
325 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 60 viral subunits form the icosahedral capsid
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: blot force = 1, blot time = 3 seconds, total blots = 1

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.4 sec. / Electron dose: 66 e/Å2 / Detector mode: INTEGRATING / Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Num. of grids imaged: 1 / Num. of real images: 1051
Image scansMovie frames/image: 45 / Used frames/image: 1-45

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Processing

EM software
IDNameVersionCategory
1Appionparticle selection
2Leginon3.1image acquisition
4ACECTF correction
5CTFFINDCTF correction
11FREALIGNinitial Euler assignment
12FREALIGNfinal Euler assignment
13FREALIGNclassification
14FREALIGN3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 120166
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107454 / Symmetry type: POINT

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