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- EMDB-24266: Cryo-EM structure of AAV True Type -

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Basic information

Entry
Database: EMDB / ID: EMD-24266
TitleCryo-EM structure of AAV True Type
Map data
Sample
  • Virus: Adeno-associated virus
    • Protein or peptide: Capsid protein VP1
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 VP1
Function and homology information
Biological speciesAdeno-associated virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsBennett AD / McKenna R
CitationJournal: J Struct Biol / Year: 2021
Title: Comparative structural, biophysical, and receptor binding study of true type and wild type AAV2.
Authors: Antonette Bennett / Joshua Hull / Nelly Jolinon / Julie Tordo / Katie Moss / Enswert Binns / Mario Mietzsch / Cathleen Hagemann / R Michael Linden / Andrea Serio / Paul Chipman / Duncan ...Authors: Antonette Bennett / Joshua Hull / Nelly Jolinon / Julie Tordo / Katie Moss / Enswert Binns / Mario Mietzsch / Cathleen Hagemann / R Michael Linden / Andrea Serio / Paul Chipman / Duncan Sousa / Felix Broecker / Peter Seeberger / Els Henckaerts / Robert McKenna / Mavis Agbandje-McKenna /
Abstract: Adeno-associated viruses (AAV) are utilized as gene transfer vectors in the treatment of monogenic disorders. A variant, rationally engineered based on natural AAV2 isolates, designated AAV-True Type ...Adeno-associated viruses (AAV) are utilized as gene transfer vectors in the treatment of monogenic disorders. A variant, rationally engineered based on natural AAV2 isolates, designated AAV-True Type (AAV-TT), is highly neurotropic compared to wild type AAV2 in vivo, and vectors based on it, are currently being evaluated for central nervous system applications. AAV-TT differs from AAV2 by 14 amino acids, including R585S and R588T, two residues previously shown to be essential for heparan sulfate binding of AAV2. The capsid structures of AAV-TT and AAV2 visualized by cryo-electron microscopy at 3.4 and 3.0 Å resolution, respectively, highlighted structural perturbations at specific amino acid differences. Differential scanning fluorimetry (DSF) performed at different pH conditions demonstrated that the melting temperature (T) of AAV2 was consistently ∼5 °C lower than AAV-TT, but both showed maximal stability at pH 5.5, corresponding to the pH in the late endosome, proposed as required for VP1u externalization to facilitate endosomal escape. Reintroduction of arginines at positions 585 and 588 in AAV-TT caused a reduction in T, demonstrating that the lack of basic amino acids at these positions are associated with capsid stability. These results provide structural and thermal annotation of AAV2/AAV-TT residue differences, that account for divergent cell binding, transduction, antigenic reactivity, and transduction of permissive tissues between the two viruses. Specifically, these data indicate that AAV-TT may not utilize a glycan receptor mediated pathway to enter cells and may have lower antigenic properties as compared to AAV2.
History
DepositionJun 19, 2021-
Header (metadata) releaseSep 29, 2021-
Map releaseSep 29, 2021-
UpdateOct 13, 2021-
Current statusOct 13, 2021Processing 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-7na6
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7na6
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24266.png

Downloads & links

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Map

FileDownload / File: emd_24266.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.978 Å
Density
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-8.57439 - 18.3176
Average (Standard dev.)-1.4624454e-09 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-220-220-220
Dimensions440440440
Spacing440440440
CellA=B=C: 430.32 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9780.9780.978
M x/y/z440440440
origin x/y/z0.0000.0000.000
length x/y/z430.320430.320430.320
α/β/γ90.00090.00090.000
start NX/NY/NZ-220-220-220
NX/NY/NZ440440440
MAP C/R/S213
start NC/NR/NS-220-220-220
NC/NR/NS440440440
D min/max/mean-8.57418.318-0.000

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Supplemental data

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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 protein VP1

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Supramolecule #1: Adeno-associated virus

SupramoleculeName: Adeno-associated virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 10804 / Sci species name: Adeno-associated virus / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes
Host systemOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: SF9
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
Molecular weightTheoretical: 58.482258 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DGVGNSSGNW HCDSTWMGDR VITTSTRTWA LPTYNNHLYK QISSQSGASN DNHYFGYSTP WGYFDFNRFH CHFSPRDWQR LINNNWGFR PKRLSFKLFN IQVKEVTQND GTTTIANNLT STVQVFTDSE YQLPYVLGSA HQGCLPPFPA DVFMVPQYGY L TLNNGSQA ...String:
DGVGNSSGNW HCDSTWMGDR VITTSTRTWA LPTYNNHLYK QISSQSGASN DNHYFGYSTP WGYFDFNRFH CHFSPRDWQR LINNNWGFR PKRLSFKLFN IQVKEVTQND GTTTIANNLT STVQVFTDSE YQLPYVLGSA HQGCLPPFPA DVFMVPQYGY L TLNNGSQA VGRSSFYCLE YFPSQMLRTG NNFTFSYTFE DVPFHSSYAH SQSLDRLMNP LIDQYLYYLS RTNTPSGTTT MS RLQFSQA GASDIRDQSR NWLPGPCYRQ QRVSKTAADN NNSDYSWTGA TKYHLNGRDS LVNPGPAMAS HKDDEEKYFP QSG VLIFGK QDSGKTNVDI EKVMITDEEE IRTTNPVATE QYGSVSTNLQ SGNTQAATSD VNTQGVLPGM VWQDRDVYLQ GPIW AKIPH TDGHFHPSPL MGGFGLKHPP PQILIKNTPV PANPSTTFSA AKFASFITQY STGQVSVEIE WELQKENSKR WNPEI QYTS NYNKSVNVDF TVDTNGVYSE PRPIGTRYLT RNL

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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
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: DIRECT ELECTRON DE-64 (8k x 8k) / Detector mode: COUNTING / Number real images: 1444 / Average electron dose: 59.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 14778
CTF correctionSoftware - Name: Auto3DEM
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: COMMON LINE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 9241

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT / Overall B value: 25
Output model

PDB-7na6:
Cryo-EM structure of AAV True Type

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