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- EMDB-22987: Adeno-associated virus serotype 5 at 2.1 Angstroms resolution, AAV5 -

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Basic information

Entry
Database: EMDB / ID: EMD-22987
TitleAdeno-associated virus serotype 5 at 2.1 Angstroms resolution, AAV5
Map data2.1A Native AAV5 virus like particle sharpened map.
Sample
  • Virus: Adeno-associated virus
    • Protein or peptide: Capsid proteinCapsid
KeywordsAAV5 / AAV / AAV-5 / Adeno Associated Virus / VIRUS LIKE PARTICLE / parvovirus / virus / gene therapy
Function / homologyPhospholipase 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 / T=1 icosahedral viral capsid / structural molecule activity / Capsid protein
Function and homology information
Biological speciesAdeno-associated virus - 5 / Adeno-associated virus
Methodsingle particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsSilveria M / Chapman MS
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122564 United States
CitationJournal: Viruses / Year: 2020
Title: The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors.
Authors: Mark A Silveria / Edward E Large / Grant M Zane / Tommi A White / Michael S Chapman /
Abstract: Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology ...Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.
History
DepositionNov 10, 2020-
Header (metadata) releaseDec 16, 2020-
Map releaseDec 16, 2020-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 25
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 25
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kp3
  • Surface level: 25
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7kp3
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22987.png

Downloads & links

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Map

FileDownload / File: emd_22987.map.gz / Format: CCP4 / Size: 729 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation2.1A Native AAV5 virus like particle sharpened map.
Voxel sizeX=Y=Z: 0.5325 Å
Density
Contour LevelBy AUTHOR: 25.0 / Movie #1: 25
Minimum - Maximum-91.554400000000001 - 152.433459999999997
Average (Standard dev.)-0.000000000898096 (±10.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-287-287-287
Dimensions576576576
Spacing576576576
CellA=B=C: 306.72003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.53250.53250.5325
M x/y/z576576576
origin x/y/z0.0000.0000.000
length x/y/z306.720306.720306.720
α/β/γ90.00090.00090.000
start NX/NY/NZ-287-287-287
NX/NY/NZ576576576
MAP C/R/S213
start NC/NR/NS-287-287-287
NC/NR/NS576576576
D min/max/mean-91.554152.433-0.000

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Supplemental data

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Mask #1

Fileemd_22987_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 2.1A Native AAV5 virus like particle unsharpened map.

Fileemd_22987_additional_1.map
Annotation2.1A Native AAV5 virus like particle unsharpened map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 2.1A Native AAV5 virus like particle sharpened map.

Fileemd_22987_half_map_1.map
Annotation2.1A Native AAV5 virus like particle sharpened map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 2.1A Native AAV5 virus like particle half map...

Fileemd_22987_half_map_2.map
Annotation2.1A Native AAV5 virus like particle half map 2 of 2 in 222a viewing frame.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Adeno-associated virus

EntireName: Adeno-associated virus
Components
  • Virus: Adeno-associated virus
    • Protein or peptide: Capsid proteinCapsid

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Supramolecule #1: Adeno-associated virus

SupramoleculeName: Adeno-associated virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Expressed using SF9 cells with a pfastbac LIC vector. Purified with cesium chloride ultracentrifugation.
NCBI-ID: 272636 / Sci species name: Adeno-associated virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.746 MDa
Virus shellShell ID: 1 / Diameter: 250.0 Å / T number (triangulation number): 1

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Macromolecule #1: Capsid protein

MacromoleculeName: Capsid protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Adeno-associated virus - 5
Molecular weightTheoretical: 80.366211 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: SFVDHPPDWL EEVGEGLREF LGLEAGPPKP KPNQQHQDQA RGLVLPGYNY LGPGNGLDRG EPVNRADEVA REHDISYNEQ LEAGDNPYL KYNHADAEFQ EKLADDTSFG GNLGKAVFQA KKRVLEPFGL VEEGAKTAPT GKRIDDHFPK RKKARTEEDS K PSTSSDAE ...String:
SFVDHPPDWL EEVGEGLREF LGLEAGPPKP KPNQQHQDQA RGLVLPGYNY LGPGNGLDRG EPVNRADEVA REHDISYNEQ LEAGDNPYL KYNHADAEFQ EKLADDTSFG GNLGKAVFQA KKRVLEPFGL VEEGAKTAPT GKRIDDHFPK RKKARTEEDS K PSTSSDAE AGPSGSQQLQ IPAQPASSLG ADTMSAGGGG PLGDNNQGAD GVGNASGDWH CDSTWMGDRV VTKSTRTWVL PS YNNHQYR EIKSGSVDGS NANAYFGYST PWGYFDFNRF HSHWSPRDWQ RLINNYWGFR PRSLRVKIFN IQVKEVTVQD STT TIANNL TSTVQVFTDD DYQLPYVVGN GTEGCLPAFP PQVFTLPQYG YATLNRDNTE NPTERSSFFC LEYFPSKMLR TGNN FEFTY NFEEVPFHSS FAPSQNLFKL ANPLVDQYLY RFVSTNNTGG VQFNKNLAGR YANTYKNWFP GPMGRTQGWN LGSGV NRAS VSAFATTNRM ELEGASYQVP PQPNGMTNNL QGSNTYALEN TMIFNSQPAN PGTTATYLEG NMLITSESET QPVNRV AYN VGGQMATNNQ SSTTAPATGT YNLQEIVPGS VWMERDVYLQ GPIWAKIPET GAHFHPSPAM GGFGLKHPPP MMLIKNT PV PGNITSFSDV PVSSFITQYS TGQVTVEMEW ELKKENSKRW NPEIQYTNNY NDPQFVDFAP DSTGEYRTTR PIGTRYLT R PL

UniProtKB: Capsid protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.75 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
50.0 mMHEPES
25.0 mMMagnesium chlorideMgCl2
25.0 mMSodium chlorideNaClSodium chloride
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
Details: Two 2uL aliquots applied to grid (manual blotting between), prior to automated 3 second blot before plunging..
DetailsMonodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -2.7 µm / Nominal defocus min: -0.7000000000000001 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 93.0 K / Max: 93.0 K
DetailsComa-free alignment and objective astigmatism where corrected using Sherpa (Thermo Fisher, Inc.).
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 1 / Number real images: 9490 / Average electron dose: 31.7 e/Å2 / Details: Pixel size was 0.5295 angstrom.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 672106
Details: LoG Picker was used for initial automated particle selection. Templates were then generated by 2D classification, followed by particle template selection in Relion 3.1 beta.
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1 beta)
Final 3D classificationSoftware - Name: RELION (ver. 3.1 beta)
Details: Multiple rounds of 2D classification and 3D classification were used to remove outliers, resulting in 373,426 particles after deduplication.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1 beta)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1 Beta) / Number images used: 373426

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Atomic model buiding 1

Initial modelPDB ID:

3ntt


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsStand-alone RSRef was used for refinement of magnification, resolution, envelope correction and atomic B-factors. This was alternated with RSRef-embedded CNS was used for molecular dynamics optimization (1st round) and stereochemically-restrained all-atom least-squares optimization.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Least-squares residual
Output model

PDB-7kp3:
Adeno-associated virus serotype 5 at 2.1 Angstroms resolution, AAV5

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