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- EMDB-24513: AAVrh.10-7x capsid -

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Basic information

Entry
Database: EMDB / ID: EMD-24513
TitleAAVrh.10-7x capsid
Map data
Sample
  • Virus: Adeno-associated virus
    • Protein or peptide: Capsid protein VP1
  • Ligand: 2'-DEOXYCYTIDINE-5'-MONOPHOSPHATE
  • Ligand: 2'-DEOXYADENOSINE-5'-MONOPHOSPHATE
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: 2.71 Å
AuthorsMietzsch M / McKenna R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM082946 United States
CitationJournal: J Virol / Year: 2021
Title: Structural Study of Aavrh.10 Receptor and Antibody Interactions.
Authors: Mario Mietzsch / Jennifer C Yu / Jane Hsi / Paul Chipman / Felix Broecker / Zhang Fuming / Robert J Linhardt / Peter H Seeberger / Regine Heilbronn / Robert McKenna / Mavis Agbandje-McKenna /
Abstract: Recombinant adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, ...Recombinant adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, the AAV vectors have to circumvent potential preexisting neutralizing host antibodies and bind to the receptors of the target cells. Both of these aspects have not been structurally analyzed for AAVrh.10. Here, cryo-electron microscopy and three-dimensional image reconstruction were used to map the binding site of sulfated -acetyllactosamine (LacNAc; previously shown to bind AAVrh.10) and a series of four monoclonal antibodies (MAbs). LacNAc was found to bind to a pocket located on the side of the 3-fold capsid protrusion that is mostly conserved to AAV9 and equivalent to its galactose-binding site. As a result, AAVrh.10 was also shown to be able to bind to cell surface glycans with terminal galactose. For the antigenic characterization, it was observed that several anti-AAV8 MAbs cross-react with AAVrh.10. The binding sites of these antibodies were mapped to the 3-fold capsid protrusions. Based on these observations, the AAVrh.10 capsid surface was engineered to create variant capsids that escape these antibodies while maintaining infectivity. Gene therapy vectors based on adeno-associated virus rhesus isolate 10 (AAVrh.10) have been used in several clinical trials to treat monogenetic diseases. However, compared to other AAV serotypes little is known about receptor binding and antigenicity of the AAVrh.10 capsid. Particularly, preexisting neutralizing antibodies against capsids are an important challenge that can hamper treatment efficiency. This study addresses both topics and identifies critical regions of the AAVrh.10 capsid for receptor and antibody binding. The insights gained were utilized to generate AAVrh.10 variants capable of evading known neutralizing antibodies. The findings of this study could further aid the utilization of AAVrh.10 vectors in clinical trials and help the approval of the subsequent biologics.
History
DepositionJul 23, 2021-
Header (metadata) releaseSep 8, 2021-
Map releaseSep 8, 2021-
UpdateNov 24, 2021-
Current statusNov 24, 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-7rl1
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7rl1
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24513.png

Downloads & links

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Map

FileDownload / File: emd_24513.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.049 Å
Density
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-7.30301 - 14.011523
Average (Standard dev.)-1.5472932e-09 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-200-200-200
Dimensions400400400
Spacing400400400
CellA=B=C: 419.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0491.0491.049
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z419.600419.600419.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ400400400
MAP C/R/S213
start NC/NR/NS-200-200-200
NC/NR/NS400400400
D min/max/mean-7.30314.012-0.000

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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
  • Ligand: 2'-DEOXYCYTIDINE-5'-MONOPHOSPHATE
  • Ligand: 2'-DEOXYADENOSINE-5'-MONOPHOSPHATE

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Supramolecule #1: Adeno-associated virus

SupramoleculeName: Adeno-associated virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1 / NCBI-ID: 272636 / Sci species name: Adeno-associated virus / Sci species strain: rh.10-7x / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No
Host systemOrganism: Homo sapiens (human) / Recombinant cell: HEK293

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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: 58.323359 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: ADGVGSSSGN WHCDSTWLGD RVITTSTRTW ALPTYNNHLY KQISNGTSGG STNDNTYFGY STPWGYFDFN RFHCHFSPRD WQRLINNNW GFRPKRLNFK LFNIQVKEVT QNEGTKTIAN NLTSTIQVFT DSEYQLPYVL GSAHQGCLPP FPADVFMIPQ Y GYLTLNNG ...String:
ADGVGSSSGN WHCDSTWLGD RVITTSTRTW ALPTYNNHLY KQISNGTSGG STNDNTYFGY STPWGYFDFN RFHCHFSPRD WQRLINNNW GFRPKRLNFK LFNIQVKEVT QNEGTKTIAN NLTSTIQVFT DSEYQLPYVL GSAHQGCLPP FPADVFMIPQ Y GYLTLNNG AQAVGRSSFY CLEYFPSQML RTGNNFEFSY QFEDVPFHSS YAHSQSLDRL MNPLIDQYLY YLSRTQTGGT AG TQQLLFS QAGPNNMSAQ AKNWLPGPCY RQQRVSTTLS QNNNSNFAWT GATKYHLNGR DSLVNPGVAM ATHKDDEERF FPS SGVLMF GKQGAGKDNV DYSAVMLTSE EEIKTTNPVA TEQYGVVADN LQQNSAQPIV GAVNSQGALP GMVWQNRDVY LQGP IWAKI PHTDGNFHPS PLMGGFGLKH PPPQILIKNT PVPADPPTTF SQAKLASFIT QYSTGQVSVE IEWELQKENS KRWNP EIQY TSNYYKSTNV DFAVNVDGTY SEPRPIGTRY LTRNL

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

MacromoleculeName: 2'-DEOXYCYTIDINE-5'-MONOPHOSPHATE / type: ligand / ID: 2 / Number of copies: 60 / Formula: DC
Molecular weightTheoretical: 307.197 Da
Chemical component information

ChemComp-DC:
2'-DEOXYCYTIDINE-5'-MONOPHOSPHATE / dCMP*YM / Deoxycytidine monophosphate

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Macromolecule #3: 2'-DEOXYADENOSINE-5'-MONOPHOSPHATE

MacromoleculeName: 2'-DEOXYADENOSINE-5'-MONOPHOSPHATE / type: ligand / ID: 3 / 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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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

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Image processing

CTF correctionSoftware - Name: cisTEM
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 17861

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