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- EMDB-0553: Cryo-EM structure of AAV-2 in complex with AAVR PKD domains 1 and 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-0553
TitleCryo-EM structure of AAV-2 in complex with AAVR PKD domains 1 and 2
Map data
SampleAdeno-associated virus - 2
  • virus
  • Binary complex of AAV-2 with a fragment of its cellular receptor, AAVR or dyslexia-associated protein KIAA0319-like protein.
  • Dyslexia-associated protein KIAA0319-like protein
  • Capsid protein VP1
  • (ligand) x 2
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 speciesAdeno-associated virus - 2 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsMeyer NL / Xie Q / Davulcu O / Yoshioka C / Chapman MS
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 ReportPDB-ID: 6nz0

SummaryFull reportAbout validation report
DateDeposition: Feb 12, 2019 / Header (metadata) release: Apr 3, 2019 / Map release: Jun 12, 2019 / Update: Jun 19, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.053
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.053
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6nz0
  • Surface level: 0.053
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6nz0
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0553.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 384 pix.
= 401.664 Å
1.05 Å/pix.
x 384 pix.
= 401.664 Å
1.05 Å/pix.
x 384 pix.
= 401.664 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.046 Å
Density
Contour LevelBy AUTHOR: 0.053 / Movie #1: 0.053
Minimum - Maximum-0.30914757 - 0.5151396
Average (Standard dev.)0.0015742391 (±0.02952454)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-192-192-192
Dimensions384384384
Spacing384384384
CellA=B=C: 401.664 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0461.0461.046
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z401.664401.664401.664
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-192-192-192
NC/NR/NS384384384
D min/max/mean-0.3090.5150.002

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Supplemental data

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Mask #1

Fileemd_0553_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Adeno-associated virus - 2

EntireName: Adeno-associated virus - 2
Details: AAV-2 virus-like particles expressed in Sf9 cells using Invitrogen's Bac-to-Bac expression system
Number of components: 6

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Component #1: virus, Adeno-associated virus - 2

VirusName: Adeno-associated virus - 2 / Class: VIRUS-LIKE PARTICLE
Details: AAV-2 virus-like particles expressed in Sf9 cells using Invitrogen's Bac-to-Bac expression system
Empty: Yes / Enveloped: No / Isolate: SEROTYPE
MassTheoretical: 22 kDa
SpeciesSpecies: Adeno-associated virus - 2
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)
Source (natural)Host Species: Homo sapiens (human)

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Component #2: protein, Binary complex of AAV-2 with a fragment of its cellular ...

ProteinName: Binary complex of AAV-2 with a fragment of its cellular receptor, AAVR or dyslexia-associated protein KIAA0319-like protein.
Details: Expressed N-terminally His6-tagged fragment of AAVR containing the PKD1 and PKD2 domains.
Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Dyslexia-associated protein KIAA0319-like protein

ProteinName: Dyslexia-associated protein KIAA0319-like protein / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 31.837385 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Capsid protein VP1

ProteinName: Capsid protein VP1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 82.077445 kDa
SourceSpecies: Adeno-associated virus - 2
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #5: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #6: ligand, water

LigandName: water / Number of Copies: 143 / Recombinant expression: No
MassTheoretical: 1.801505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.1 mg/mL / pH: 7.4
Support filmGlow discharge settings: Pelco easiGlow, 25s, 25mA, 0.39mBar, negative polarity Grid: Ted Pella Cat#01824, ultrathin carbon film (<3nm) on lacey carbon support film
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 298.15 K / Humidity: 100 %
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
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25.4 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 75000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: -800.0 - -2000.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON III (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2329

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 21373
3D reconstructionSoftware: RELION / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible
Target criteria: Map values at grid points within 2 Angstrom of atoms
Refinement space: REAL
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.
Input PDB model: 1LP3
Chain ID: A
Output model

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