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- PDB-3j4p: Electron Microscopy Analysis of a Disaccharide Analog complex Rev... -

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Entry
Database: PDB / ID: 3j4p
TitleElectron Microscopy Analysis of a Disaccharide Analog complex Reveals Receptor Interactions of Adeno-Associated Virus
ComponentsCapsid protein VP1
KeywordsVIRUS / Virus cell-receptor interaction
Function / homology
Function and homology information


symbiont entry into host cell via permeabilization of host membrane / host cell nucleolus / T=1 icosahedral viral capsid / clathrin-dependent endocytosis of virus by host cell / virion attachment to host cell / structural molecule activity
Similarity search - Function
Phospholipase 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
Similarity search - Domain/homology
sucrose octasulfate / Capsid protein VP1
Similarity search - Component
Biological speciesAdeno-associated virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsXie, Q. / Chapman, M.S.
CitationJournal: J Struct Biol / Year: 2013
Title: Electron microscopy analysis of a disaccharide analog complex reveals receptor interactions of adeno-associated virus.
Authors: Qing Xie / Michael Spilman / Nancy L Meyer / Thomas F Lerch / Scott M Stagg / Michael S Chapman /
Abstract: Mechanistic studies of macromolecular complexes often feature X-ray structures of complexes with bound ligands. The attachment of adeno-associated virus (AAV) to cell surface glycosaminoglycans (GAGs) ...Mechanistic studies of macromolecular complexes often feature X-ray structures of complexes with bound ligands. The attachment of adeno-associated virus (AAV) to cell surface glycosaminoglycans (GAGs) is an example that has not proven amenable to crystallography, because the binding of GAG analogs disrupts lattice contacts. The interactions of AAV with GAGs are of interest in mediating the cell specificity of AAV-based gene therapy vectors. Previous electron microscopy led to differing conclusions on the exact binding site and the existence of large ligand-induced conformational changes in the virus. Conformational changes are expected during cell entry, but it has remained unclear whether the electron microscopy provided evidence of their induction by GAG-binding. Taking advantage of automated data collection, careful processing and new methods of structure refinement, the structure of AAV-DJ complexed with sucrose octasulfate is determined by electron microscopy difference map analysis to 4.8Å resolution. At this higher resolution, individual sulfate groups are discernible, providing a stereochemical validation of map interpretation, and highlighting interactions with two surface arginines that have been implicated in genetic studies. Conformational changes induced by the SOS are modest and limited to the loop most directly interacting with the ligand. While the resolution attainable will depend on sample order and other factors, there are an increasing number of macromolecular complexes that can be studied by cryo-electron microscopy at resolutions beyond 5Å, for which the approaches used here could be used to characterize the binding of inhibitors and other small molecule effectors when crystallography is not tractable.
History
DepositionSep 10, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 16, 2013Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2013Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name
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_conn_type / 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.type / _entity.formula_weight / _entity.pdbx_description / _entity.type
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Feb 21, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_oper_list
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _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)59,6084
Polymers58,5781
Non-polymers1,0303
Water181
1
A: Capsid protein VP1
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)3,576,458240
Polymers3,514,65260
Non-polymers61,806180
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
  • 298 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)298,03820
Polymers292,8885
Non-polymers5,15015
Water905
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Capsid protein VP1
hetero molecules
x 6


  • icosahedral 23 hexamer
  • 358 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)357,64624
Polymers351,4656
Non-polymers6,18118
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 / Fragment: SEE REMARK 999
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus / Gene: cap / Plasmid: pAVDJ / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): sf9 / References: UniProt: P03135*PLUS
#2: Polysaccharide 1,3,4,6-tetra-O-sulfo-beta-D-fructofuranose-(2-1)-2,3,4,6-tetra-O-sulfonato-alpha-D-glucopyranose / sucrose octasulfate


Type: oligosaccharide, Oligosaccharide / Class: Substrate analog / Mass: 982.803 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide with reducing-end-to-reducing-end glycosidic bond
References: sucrose octasulfate
DescriptorTypeProgram
WURCS=2.0/2,2,1/[ha122h-2b_2-5_1*OSO/3=O/3=O_3*OSO/3=O/3=O_4*OSO/3=O/3=O_6*OSO/3=O/3=O][a2122h-1a_1-5_2*OSO/3=O/3=O_3*OSO/3=O/3=O_4*OSO/3=O/3=O_6*OSO/3=O/3=O]/1-2/a2-b1WURCSPDB2Glycan 1.1.0
[][b-D-Fruf1SO33SO34SO36SO3]{[(2+1)][a-D-Glcp2SO33SO34SO36SO3]{}}LINUCSPDB-CARE
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsSAMPLE CONTAINED FULL-LENGTH CAPSID PROTEIN, BUT RESIDUES 1-220 WERE NOT MODELED.

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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: Recombinant Adeno-Associated Virus DJ / Type: VIRUS
Molecular weightValue: 3.7 MDa / Experimental value: NO
Buffer solutionName: 10 mM Tris, 125 mM NaCl, 1 mM MgCl2 / pH: 6.8 / Details: 10 mM Tris, 125 mM NaCl, 1 mM MgCl2
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400 mesh carbon-coated grids (C-flat) glow-discharged for 5 seconds (Gatan Model 950)
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %
Details: Both sides of the grid were blotted for 2.5 seconds before plunging in liquid ethane (FEI VITROBOT MARK IV).
Method: Both sides of the grid were blotted for 2.5 seconds.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Apr 15, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 120000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Specimen holder type: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 94 K
Image recordingElectron dose: 15 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNum. digital images: 5207
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2Appion3D reconstruction
3FREALIGN3D reconstruction
CTF correctionDetails: each particle
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: Fourier methods / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45000 / Nominal pixel size: 1.3067 Å / Actual pixel size: 1.3067 Å / Details: Final map was amplitude corrected using embfactor. / Symmetry type: POINT
Atomic model buildingB value: 25 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: CC / Details: REFINEMENT PROTOCOL--RIGID BODY
Atomic model buildingPDB-ID: 3J1Q

3j1q


Accession code: 3J1Q / Source name: PDB / Type: experimental model
RefinementHighest resolution: 4.8 Å
Refinement stepCycle: LAST / Highest resolution: 4.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4137 0 57 1 4195

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