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- EMDB-20610: Atomic-Resolution Cryo-EM Structure of AAV2 VLP -

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Basic information

Entry
Database: EMDB / ID: EMD-20610
TitleAtomic-Resolution Cryo-EM Structure of AAV2 VLP
Map dataAAV2 VLP
Sample
  • Virus: Adeno-associated virus - 2
    • Protein or peptide: Capsid protein VP1
KeywordsProtrusion / axes / receptor / pore / VIRUS LIKE PARTICLE
Function / homology
Function and homology information


symbiont entry into host cell via permeabilization of host 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: 3.02 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-6u0v
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6u0v
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20610.png

Downloads & links

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Map

FileDownload / File: emd_20610.map.gz / Format: CCP4 / Size: 264.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAAV2 VLP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.91 Å/pix.
x 411 pix.
= 374.01 Å		0.91 Å/pix.
x 411 pix.
= 374.01 Å		0.91 Å/pix.
x 411 pix.
= 374.01 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 2.0 / Movie #1: 2
Minimum - Maximum-7.36091 - 14.983236
Average (Standard dev.)-0.000000000087163 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-205-205-205
Dimensions411411411
Spacing411411411
CellA=B=C: 374.01 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.910.910.91
M x/y/z411411411
origin x/y/z0.0000.0000.000
length x/y/z374.010374.010374.010
α/β/γ90.00090.00090.000
start NX/NY/NZ-205-205-205
NX/NY/NZ411411411
MAP C/R/S213
start NC/NR/NS-205-205-205
NC/NR/NS411411411
D min/max/mean-7.36114.983-0.000

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Supplemental data

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Sample components

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Entire : Adeno-associated virus - 2

EntireName: Adeno-associated virus - 2
Components
  • Virus: Adeno-associated virus - 2
    • Protein or peptide: Capsid protein VP1

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Supramolecule #1: Adeno-associated virus - 2

SupramoleculeName: Adeno-associated virus - 2 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 10804 / Sci species name: Adeno-associated virus - 2 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: Yes
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: 59.129949 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: NNEGADGVGN SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ SGASNDNHYF GYSTPWGYFD FNRFHCHFSP RDWQRLINN NWGFRPKRLN FKLFNIQVKE VTQNDGTTTI ANNLTSTVQV FTDSEYQLPY VLGSAHQGCL PPFPADVFMV P QYGYLTLN ...String:
NNEGADGVGN SSGNWHCDST WMGDRVITTS TRTWALPTYN NHLYKQISSQ SGASNDNHYF GYSTPWGYFD FNRFHCHFSP RDWQRLINN NWGFRPKRLN FKLFNIQVKE VTQNDGTTTI ANNLTSTVQV FTDSEYQLPY VLGSAHQGCL PPFPADVFMV P QYGYLTLN NGSQAVGRSS FYCLEYFPSQ MLRTGNNFTF SYTFEDVPFH SSYAHSQSLD RLMNPLIDQY LYYLSRTNTP SG TTTQSRL QFSQAGASDI RDQSRNWLPG PCYRQQRVSK TSADNNNSEY SWTGATKYHL NGRDSLVNPG PAMASHKDDE EKF FPQSGV LIFGKQGSEK TNVDIEKVMI TDEEEIRTTN PVATEQYGSV STNLQRGNRQ AATADVNTQG VLPGMVWQDR DVYL QGPIW AKIPHTDGHF HPSPLMGGFG LKHPPPQILI KNTPVPANPS TTFSAAKFAS FITQYSTGQV SVEIEWELQK ENSKR WNPE IQYTSNYNKS VNVDFTVDTN GVYSEPRPIG TRYLTRNL

UniProtKB: Capsid protein VP1

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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
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 13557
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Auto3DEM / Number images used: 6778
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: COMMON LINE

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Atomic model buiding 1

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

PDB-6u0v:
Atomic-Resolution Cryo-EM Structure of AAV2 VLP

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