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- PDB-6nz0: Cryo-EM structure of AAV-2 in complex with AAVR PKD domains 1 and 2 -

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Basic information

Entry
Database: PDB / ID: 6nz0
TitleCryo-EM structure of AAV-2 in complex with AAVR PKD domains 1 and 2
Components
  • Capsid protein VP1
  • Dyslexia-associated protein KIAA0319-like protein
KeywordsVIRUS / AAV / AAVR / receptor / coreceptor / KIAA0319L / PKD / parvovirus
Function / homology
Function and homology information


T=1 icosahedral viral capsid / permeabilization of host organelle membrane involved in viral entry into host cell / viral entry via permeabilization of inner membrane / host cell nucleolus / cytoplasmic vesicle / clathrin-dependent endocytosis of virus by host cell / virion attachment to host cell / Golgi membrane / structural molecule activity / integral component of membrane / plasma membrane
Parvovirus coat protein VP1, N-terminal / PKD domain / PKD domain superfamily / Dyslexia-associated protein KIAA0319-like protein / PKD/Chitinase domain / Capsid/spike protein, ssDNA virus / MANSC domain / Immunoglobulin-like fold / Parvovirus coat protein VP1 / Parvovirus coat protein VP2 ...Parvovirus coat protein VP1, N-terminal / PKD domain / PKD domain superfamily / Dyslexia-associated protein KIAA0319-like protein / PKD/Chitinase domain / Capsid/spike protein, ssDNA virus / MANSC domain / Immunoglobulin-like fold / Parvovirus coat protein VP1 / Parvovirus coat protein VP2 / Parvovirus coat protein VP1/VP2 / Parvovirus coat protein VP2 / Polycystic kidney disease (PKD) domain profile. / MANSC domain profile.
Capsid protein VP1 / Dyslexia-associated protein KIAA0319-like protein
Biological speciesHomo sapiens (human)
Adeno-associated virus - 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsMeyer, N.L. / Xie, Q. / Davulcu, O. / Yoshioka, C. / Chapman, M.S.
Funding supportUnited States , 2件
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR35GM122564United States
National Institutes of Health/National Institute of General Medical SciencesR01GM066875United States
CitationJournal: Elife / Year: 2019
Title: Structure of the gene therapy vector, adeno-associated virus with its cell receptor, AAVR.
Authors: Nancy L Meyer / Guiqing Hu / Omar Davulcu / Qing Xie / Alex J Noble / Craig Yoshioka / Drew S Gingerich / Andrew Trzynka / Larry David / Scott M Stagg / Michael Stewart Chapman /
Abstract: Adeno-associated virus (AAV) vectors are preeminent in emerging clinical gene therapies. Generalizing beyond the most tractable genetic diseases will require modulation of cell specificity and immune ...Adeno-associated virus (AAV) vectors are preeminent in emerging clinical gene therapies. Generalizing beyond the most tractable genetic diseases will require modulation of cell specificity and immune neutralization. Interactions of AAV with its cellular receptor, AAVR, are key to understanding cell-entry and trafficking with the rigor needed to engineer tissue-specific vectors. -electron tomography shows ordered binding of part of the flexible receptor to the viral surface, with distal domains in multiple conformations. Regions of the virus and receptor in close physical proximity can be identified by cross-linking/mass spectrometry. -electron microscopy with a two-domain receptor fragment reveals the interactions at 2.4 Å resolution. AAVR binds between AAV's spikes on a plateau that is conserved, except in one clade whose structure is AAVR-incompatible. AAVR's footprint overlaps the epitopes of several neutralizing antibodies, prompting a re-evaluation of neutralization mechanisms. The structure provides a roadmap for experimental probing and manipulation of viral-receptor interactions.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 12, 2019 / Release: Jun 12, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jun 12, 2019Structure modelrepositoryInitial release
1.1Jun 19, 2019Structure modelData collection / Database referencescitation / citation_author_citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID

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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
Z: Dyslexia-associated protein KIAA0319-like protein
A: Capsid protein VP1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,9393
Polymers113,9152
Non-polymers241
Water2,576143
1
Z: Dyslexia-associated protein KIAA0319-like protein
A: Capsid protein VP1
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)6,836,348180
Polymers6,834,890120
Non-polymers1,45860
Water1,08160
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
Z: Dyslexia-associated protein KIAA0319-like protein
A: Capsid protein VP1
hetero molecules
x 5


  • icosahedral pentamer
  • 570 kDa, 10 polymers
Theoretical massNumber of molelcules
Total (without water)569,69615
Polymers569,57410
Non-polymers1225
Water905
TypeNameSymmetry operationNumber
point symmetry operation5
4
Z: Dyslexia-associated protein KIAA0319-like protein
A: Capsid protein VP1
hetero molecules
x 6


  • icosahedral 23 hexamer
  • 684 kDa, 12 polymers
Theoretical massNumber of molelcules
Total (without water)683,63518
Polymers683,48912
Non-polymers1466
Water1086
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: Protein/peptide Dyslexia-associated protein KIAA0319-like protein / Adeno-associated virus receptor / AAVR


Mass: 31837.385 Da / Num. of mol.: 1 / Fragment: residues 311-597
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KIAA0319L, AAVR, KIAA1837, PP791 / Plasmid: pET-11a / Production host: Escherichia coli (E. coli) / Variant (production host): NEBexpress / References: UniProt: Q8IZA0
#2: Protein/peptide Capsid protein VP1 /


Mass: 82077.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus - 2 / Gene: VP1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P03135
#3: Chemical ChemComp-MG / MAGNESIUM ION


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


Mass: 18.015 Da / Num. of mol.: 143 / Source method: isolated from a natural source / Formula: H2O / Water

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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

Source: RECOMBINANT

IDNameTypeDetailsEntity IDParent-ID
1Adeno-associated virus - 2VIRUSAAV-2 virus-like particles expressed in Sf9 cells using Invitrogen's Bac-to-Bac expression system1,20
2Binary complex of AAV-2 with a fragment of its cellular receptor, AAVR or dyslexia-associated protein KIAA0319-like protein.COMPLEXExpressed N-terminally His6-tagged fragment of AAVR containing the PKD1 and PKD2 domains.11
Molecular weight

Entity assembly-ID: 1

IDValue (°)Experimental value
13.8 MDaNO
20.022 MDa
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Adeno-associated virus - 210804
31Adeno-associated virus - 210804
42Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
31Sf9562
42Escherichia coli (E. coli)562
Details of virus
IDEntity assembly-IDEmptyEnvelopedIsolateType
11YESNOSEROTYPEVIRUS-LIKE PARTICLE
22
Natural host

ID: 1

Entity assembly-IDOrganismNcbi tax-ID
1Homo sapiens9606
2
Virus shell

ID: 1

Entity assembly-IDDiameter (nm)Triangulation number (T number)
12501
2
Buffer solutionpH: 7.4
Buffer component

Buffer-ID: 1

IDConc.Name
125 mMHEPES
2150 mMNaClSodium chloride
350 mMMgCl2
SpecimenConc.: 0.1 mg/ml
Details: Virus-like particles at 0.10 mg/mL were adhered to thin continuous carbon-coated grids, then, after initial blotting, AAVR-PKD1-2 was added at 0.74 mg/mL (a 10-fold molar excess over AAV2 subunits) before blotting again.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Glow discharge settings: Pelco easiGlow, 25s, 25mA, 0.39mBar, negative polarity Grid: Ted Pella Cat#01824, ultrathin carbon film (<3nm) on lacey carbon support film
Grid material: COPPER / Grid mesh size: 400 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298.15 K
Details: 4ul of AAV-2 (1.7uM) was added to the grid, followed by 4ul of AAVR-PKD1-2 (16.7uM), with manual blotting after each addition with Whatman paper (Cat. no. 1001-110.) 4ul of buffer containing 25mM HEPES, 150mM NaCl, pH 7.4 was added to the grid before final blotting and plunge-freezing in the Vitrobot (blot time 1.5sec, blot force -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: 75000 X / Nominal defocus max: -2000 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 40 sec. / Electron dose: 25.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2329
Image scansWidth: 7676 / Height: 7420

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.0bparticle selection
2SerialEMimage acquisition
4Gctf1.06CTF correctionresolution range: 30-3A
7Cootmodel fitting
9RELION3.0binitial Euler assignment
10RELION3.0bfinal Euler assignment
11RELION3.0bclassification
12RELION3.0b3D reconstruction
19RSRefmodel refinementhttps://biochem.missouri.edu/chapman/software.htm
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 34450
Details: RELION Autopicker using 3D AAV-2 reference filtered to 20A with 15 degree sampling
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21373 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Target criteria: Map values at grid points within 2 Angstrom of atoms
Details: An initial model for AAVR was constructed by homology modeling using Modeller 9.2. After remodeling AAVR and the crystal structure of AAV2, the structure was refined using RSRef embedded in CNS for stereochemically restrained torsion angle simulated annealing and gradient descent optimization. Stand-alone RSRef was used for refinement of EM envelope corrections, resolution estimates, and restrained atomic B-factors.
Atomic model buildingPDB-ID: 1LP3
Pdb chain-ID: A / Pdb chain residue range: 217-735

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