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- EMDB-20609: Cryo-EM structure of the chimeric vector AAV2.7m8 -

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Basic information

Entry
Database: EMDB / ID: EMD-20609
TitleCryo-EM structure of the chimeric vector AAV2.7m8
Map datachimeric vector AAV2.7m8
SampleAdeno-associated virus - 2.7m8 != Adeno-associated virus - 2

Adeno-associated virus - 2.7m8

  • Virus: Adeno-associated virus - 2
    • Protein or peptide: Capsid protein VP1
  • Ligand: 2'-DEOXYADENOSINE-5'-MONOPHOSPHATE
KeywordsProtrusion / insertion / receptor / VIRUS
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 speciesAdeno-associated virus - 2
Methodsingle particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsAgbandje-McKenna M / Bennett A
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R01 GM109524 United States
CitationJournal: J Struct Biol / Year: 2020
Title: Structure comparison of the chimeric AAV2.7m8 vector with parental AAV2.
Authors: Antonette Bennett / Annahita Keravala / Victoria Makal / Justin Kurian / Brahim Belbellaa / Rangoli Aeran / Yu-Shan Tseng / Duncan Sousa / John Spear / Mehdi Gasmi / Mavis Agbandje-McKenna /
Abstract: The AAV2.7m8 vector is an engineered capsid with a 10-amino acid insertion in adeno-associated virus (AAV) surface variable region VIII (VR-VIII) resulting in the alteration of an antigenic region of ...The AAV2.7m8 vector is an engineered capsid with a 10-amino acid insertion in adeno-associated virus (AAV) surface variable region VIII (VR-VIII) resulting in the alteration of an antigenic region of AAV2 and the ability to efficiently transduce retina cells following intravitreal administration. Directed evolution and in vivo screening in the mouse retina isolated this vector. In the present study, we sought to identify the structural differences between a recombinant AAV2.7m8 (rAAV2.7m8) vector packaging a GFP genome and its parental serotype, AAV2, by cryo-electron microscopy (cryo-EM) and image reconstruction. The structures of rAAV2.7m8 and AAV2 were determined to 2.91 and 3.02 Å resolution, respectively. The rAAV2.7m8 amino acid side-chains for residues 219-745 (the last C-terminal residue) were interpretable in the density map with the exception of the 10 inserted amino acids. While observable in a low sigma threshold density, side-chains were only resolved at the base of the insertion, likely due to flexibility at the top of the loop. A comparison to parental AAV2 (ordered from residues 217-735) showed the structures to be similar, except at some side-chains that had different orientations and, in VR-VIII containing the 10 amino acid insertion. VR-VIII is part of an AAV2 antigenic epitope, and the difference is consistent with rAAV2.7m8's escape from a known AAV2 monoclonal antibody, C37-B. The observations provide valuable insight into the configuration of inserted surface peptides on the AAV capsid and structural differences to be leveraged for future AAV vector rational design, especially for retargeted tropism and antibody escape.
History
DepositionAug 14, 2019-
Header (metadata) releaseSep 18, 2019-
Map releaseAug 19, 2020-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6u0r
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6u0r
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20609.png

Downloads & links

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Map

FileDownload / File: emd_20609.map.gz / Format: CCP4 / Size: 284.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationchimeric vector AAV2.7m8
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy EMDB: 2.0 / Movie #1: 2
Minimum - Maximum-10.619336000000001 - 18.651861
Average (Standard dev.)-0.000000000967549 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-210-210-210
Dimensions421421421
Spacing421421421
CellA=B=C: 446.25998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z421421421
origin x/y/z0.0000.0000.000
length x/y/z446.260446.260446.260
α/β/γ90.00090.00090.000
start NX/NY/NZ-210-210-210
NX/NY/NZ421421421
MAP C/R/S213
start NC/NR/NS-210-210-210
NC/NR/NS421421421
D min/max/mean-10.61918.652-0.000

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Supplemental data

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Sample components

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Entire : Adeno-associated virus - 2.7m8

EntireName: Adeno-associated virus - 2.7m8
Components
  • Virus: Adeno-associated virus - 2
    • Protein or peptide: Capsid protein VP1
  • Ligand: 2'-DEOXYADENOSINE-5'-MONOPHOSPHATE

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Supramolecule #1: Adeno-associated virus - 2

SupramoleculeName: Adeno-associated virus - 2 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1 / NCBI-ID: 10804 / Sci species name: Adeno-associated virus - 2 / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No
Molecular weightTheoretical: 4 MDa
Virus shellShell ID: 1 / Diameter: 260.0 Å / T number (triangulation number): 1

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Macromolecule #1: Capsid protein VP1

MacromoleculeName: Capsid protein VP1 / type: protein_or_peptide / ID: 1 / Number of copies: 60 / Enantiomer: LEVO
Source (natural)Organism: Adeno-associated virus - 2
Molecular weightTheoretical: 59.655652 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DGVGNSSGNW HCDSTWMGDR VITTSTRTWA LPTYNNHLYK QISSQSGASN DNHYFGYSTP WGYFDFNRFH CHFSPRDWQR LINNNWGFR PKRLNFKLFN IQVKEVTQND GTTTIANNLT STVQVFTDSE YQLPYVLGSA HQGCLPPFPA DVFMVPQYGY L TLNNGSQA ...String:
DGVGNSSGNW HCDSTWMGDR VITTSTRTWA LPTYNNHLYK QISSQSGASN DNHYFGYSTP WGYFDFNRFH CHFSPRDWQR LINNNWGFR PKRLNFKLFN IQVKEVTQND GTTTIANNLT STVQVFTDSE YQLPYVLGSA HQGCLPPFPA DVFMVPQYGY L TLNNGSQA VGRSSFYCLE YFPSQMLRTG NNFTFSYTFE DVPFHSSYAH SQSLDRLMNP LIDQYLYYLS RTNTPSGTTT QS RLQFSQA GASDIRDQSR NWLPGPCYRQ QRVSKTSADN NNSEYSWTGA TKYHLNGRDS LVNPGPAMAS HKDDEEKFFP QSG VLIFGK QGSEKTNVDI EKVMITDEEE IRTTNPVATE QYGSVSTNLQ RGNLALGETT RPARQAATAD VNTQGVLPGM VWQD RDVYL QGPIWAKIPH TDGHFHPSPL MGGFGLKHPP PQILIKNTPV PANPSTTFSA AKFASFITQY STGQVSVEIE WELQK ENSK RWNPEIQYTS NYNKSVNVDF TVDTNGVYSE PRPIGTRYLT RNL

UniProtKB: Capsid protein VP1

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Macromolecule #2: 2'-DEOXYADENOSINE-5'-MONOPHOSPHATE

MacromoleculeName: 2'-DEOXYADENOSINE-5'-MONOPHOSPHATE / type: ligand / ID: 2 / Number of copies: 60 / Formula: DA
Molecular weightTheoretical: 331.222 Da
Chemical component information

ChemComp-D5M:
2'-DEOXYADENOSINE-5'-MONOPHOSPHATE / Deoxyadenosine monophosphate

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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

Concentration1 mg/mL
BufferpH: 7.4
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 410 / Average electron dose: 75.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 27365
Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: COMMON LINE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Auto3DEM / Number images used: 27364

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT / Overall B value: 100
Output model

PDB-6u0r:
Cryo-EM structure of the chimeric vector AAV2.7m8

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