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- PDB-3j1s: Structure of adeno-associated virus-2 in complex with neutralizin... -

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Basic information

Entry
Database: PDB / ID: 3j1s
TitleStructure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20
Components
  • A20 heavy chain
  • A20 light chain
  • Capsid protein VP1
KeywordsVIRUS/IMMUNE SYSTEM / Epitope / Fab / gene therapy / VIRUS-IMMUNE SYSTEM complex
Function / homology
Function and homology information


permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner 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
Biological speciesMus musculus (house mouse)
Adeno-associated virus - 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.5 Å
AuthorsChapman, M.S. / McCraw, D.M.
CitationJournal: Virology
Title: Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20.
Authors: Dustin M McCraw / Jason K O'Donnell / Kenneth A Taylor / Scott M Stagg / Michael S Chapman /
Abstract: The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5Å resolution is determined for ...The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5Å resolution is determined for a complex of AAV-2 with the Fab' fragment of monoclonal antibody (MAb) A20, the most extensively characterized AAV MAb. The binding footprint is determined through fitting the cryo-EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. The footprint extends from the previously implicated plateau to the side of the spike, and into the conserved canyon, covering a larger area than anticipated. Comparison with structures of binding and non-binding serotypes indicates that recognition depends on a combination of subtle serotype-specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune-resistant vectors that can still bind to target cells.
History
DepositionMay 23, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 6, 2012Provider: repository / Type: Initial release
Revision 1.1Jun 20, 2012Group: Database references
Revision 1.2Jul 4, 2012Group: Database references
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id

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Structure visualization

Movie
  • 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
  • EMDB-5424
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  • Superimposition on EM map
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
L: A20 light chain
H: A20 heavy chain
A: Capsid protein VP1


Theoretical massNumber of molelcules
Total (without water)106,1543
Polymers106,1543
Non-polymers00
Water0
1
L: A20 light chain
H: A20 heavy chain
A: Capsid protein VP1
x 60


Theoretical massNumber of molelcules
Total (without water)6,369,234180
Polymers6,369,234180
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
L: A20 light chain
H: A20 heavy chain
A: Capsid protein VP1
x 5


  • icosahedral pentamer
  • 531 kDa, 15 polymers
Theoretical massNumber of molelcules
Total (without water)530,77015
Polymers530,77015
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
L: A20 light chain
H: A20 heavy chain
A: Capsid protein VP1
x 6


  • icosahedral 23 hexamer
  • 637 kDa, 18 polymers
Theoretical massNumber of molelcules
Total (without water)636,92318
Polymers636,92318
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
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: Antibody A20 light chain


Mass: 23603.965 Da / Num. of mol.: 1 / Fragment: Fab' / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)
#2: Antibody A20 heavy chain


Mass: 23777.314 Da / Num. of mol.: 1 / Fragment: Fab' / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)
#3: Protein Capsid protein VP1 / / Coat protein VP1


Mass: 58772.625 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999 / Source method: isolated from a natural source / Source: (natural) Adeno-associated virus - 2 / References: UniProt: P03135
Sequence detailsDUE TO IN-FRAME ALTERNATIVE SPLICING, THE AAV-2 CAPSID PROTEIN IN THIS ENTRY IS A 1:1:10 MIXTURE OF ...DUE TO IN-FRAME ALTERNATIVE SPLICING, THE AAV-2 CAPSID PROTEIN IN THIS ENTRY IS A 1:1:10 MIXTURE OF ISOFORMS VP1, VP2, AND VP3. THE SEQUENCE MODELED (UNP RESIDUES 217-735) IS COMMON TO ALL THREE ISOFORMS. THE VARIABLE DOMAINS OF FAB' A20 (RESIDUES 1-107 OF THE LIGHT CHAIN AND RESIDUES 1-120 OF THE HEAVY CHAIN) ARE A HOMOLOGY MODEL DERIVED FROM SEQUENCING INFORMATION. THE CONSERVED CONSTANT DOMAINS (RESIDUES 108-214 OF THE LIGHT CHAIN AND RESIDUES 121-218 OF THE HEAVY CHAIN) ARE FROM PDB ENTRY 1OSP AND ARE AN APPROXIMATION OF THE ACTUAL SEQUENCE OF FAB' A20.

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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

Component
IDNameTypeDetailsParent-IDSource
1Adeno-associated virus-2 in complex with monoclonal antibody A20COMPLEX60 A20 Fab's bind to one adeno-associated virus (one adeno-associated virus consists of 60 viral proteins). Infectious DNA-containing particle (DNA not resolved)0MULTIPLE SOURCES
2Antibody Fab' fragmentCOMPLEXFab' fragment generated by pepsin cleavage of A20 IgG antibody.1NATURAL
3Antibody Fab' fragmentCOMPLEXFab' fragment generated by pepsin cleavage of A20 IgG antibody.1NATURAL
4Viral proteinVIRUSViral capsid protein monomer for AAV-21NATURAL
Molecular weightValue: 6.9 MDa / Experimental value: NO
Details of virusDetails: Single-stranded DNA Parvovirus / Empty: NO / Enveloped: NO / Host category: VERTEBRATES / Isolate: SEROTYPE / Type: VIRION
Natural hostOrganism: Homo sapiens / Strain: HeLa
Buffer solutionName: 100 mM HEPES, 50 mM magnesium chloride, 5% glycerol / pH: 7.2
Details: 100 mM HEPES, 50 mM magnesium chloride, 5% glycerol
SpecimenConc.: 0.14 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 100 mM HEPES, 50 mM magnesium chloride, 5% glycerol
Specimen supportDetails: 400 mesh carbon grid with holey carbon support
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temp: 90 K / Humidity: 100 %
Details: Blot for 2.0 seconds before plunging into liquid ethane (FEI Vitrobot Mark IV).
Method: Blot for 2.0 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Feb 23, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: SPOT SCAN / Electron beam tilt params: 0
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 37000 X / Calibrated magnification: 39775 X / Nominal defocus max: -4000 nm / Nominal defocus min: -500 nm / Cs: 2.7 mm
Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
Camera length: 0 mm
Specimen holderSpecimen holder model: OTHER / Specimen holder type: Nitrogen cooled / Temperature: 93 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 15 e/Å2 / Film or detector model: GENERIC GATAN
Image scansNum. digital images: 1503

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Processing

EM software
IDNameCategory
1ACECTF correction
2RSRefmodel fitting
3Appion3D reconstruction
4EMAN3D reconstruction
CTF correctionDetails: Whole image
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: Common-lines / Resolution: 8.5 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 11898 / Nominal pixel size: 2.45 Å / Actual pixel size: 2.45 Å
Details: A20 FAB' WAS MODELED AS THE COMBINATION OF A HOMOLOGY MODEL AND SEQUENCE SEGMENTS EXCERPTED FROM PDB ENTRY 1OSP (SEE REMARK 999 FOR DETAILS). THE AUTHORS STATE THAT ANY INCORRECT PEPTIDE ...Details: A20 FAB' WAS MODELED AS THE COMBINATION OF A HOMOLOGY MODEL AND SEQUENCE SEGMENTS EXCERPTED FROM PDB ENTRY 1OSP (SEE REMARK 999 FOR DETAILS). THE AUTHORS STATE THAT ANY INCORRECT PEPTIDE BOND LENGTHS IN THIS ENTRY ARE THE RESULT OF RIGID-BODY FITTING TO A LOW-RESOLUTION MAP OR ARE INHERITED DIRECTLY FROM PDB ENTRY 1OSP.
Num. of class averages: 304 / Symmetry type: POINT
Atomic model building
IDProtocolSpaceTarget criteriaDetails
1RIGID BODY FITREALLeast-squares difference between experimental & model coulombic potentialMETHOD--Rigid Body REFINEMENT PROTOCOL--Rigid Body DETAILS--Anti-bumping restraints from CNS, Constrained icosahedral symmetry
2RIGID BODY FITREALLeast-squares difference between experimental & model coulombic potentialMETHOD--Rigid Body REFINEMENT PROTOCOL--Rigid Body DETAILS--Anti-bumping restraints from CNS, Constrained icosahedral symmetry
3RIGID BODY FITREALLeast-squares difference between experimental & model coulombic potentialMETHOD--Superimposed according to icosahedral symmetry REFINEMENT PROTOCOL--Fixed DETAILS--Anti-bumping restraints from CNS included 1LP3, Constrained icosahedral symmetry
Atomic model buildingPDB-ID: 1LP3

1lp3


Pdb chain-ID: A
RefinementDetails: A20 FAB' WAS MODELED AS THE COMBINATION OF A HOMOLOGY MODEL AND SEQUENCE SEGMENTS EXCERPTED FROM PDB ENTRY 1OSP (SEE REMARK 999 FOR DETAILS). THE AUTHORS STATE THAT ANY INCORRECT PEPTIDE ...Details: A20 FAB' WAS MODELED AS THE COMBINATION OF A HOMOLOGY MODEL AND SEQUENCE SEGMENTS EXCERPTED FROM PDB ENTRY 1OSP (SEE REMARK 999 FOR DETAILS). THE AUTHORS STATE THAT ANY INCORRECT PEPTIDE BOND LENGTHS IN THIS ENTRY ARE THE RESULT OF RIGID-BODY FITTING TO A LOW-RESOLUTION MAP OR ARE INHERITED DIRECTLY FROM PDB ENTRY 1OSP.
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms7485 0 0 0 7485

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