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- EMDB-26390: SAAV pH 7.4 capsid structure -

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Basic information

Entry
Database: EMDB / ID: EMD-26390
TitleSAAV pH 7.4 capsid structure
Map data
Sample
  • Virus: Snake adeno-associated virus
    • Protein or peptide: Capsid protein
KeywordsCapsid / AAV / gene therapy / receptor / endosomal trafficking / antigenicity / VIRUS LIKE PARTICLE
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 speciesSnake adeno-associated virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.25 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: 2022
Title: Characterization of the Serpentine Adeno-Associated Virus (SAAV) Capsid Structure: Receptor Interactions and Antigenicity.
Authors: Mario Mietzsch / Joshua A Hull / Victoria E Makal / Alberto Jimenez Ybargollin / Jennifer C Yu / Kedrick McKissock / Antonette Bennett / Judit Penzes / Bridget Lins-Austin / Qian Yu / Paul ...Authors: Mario Mietzsch / Joshua A Hull / Victoria E Makal / Alberto Jimenez Ybargollin / Jennifer C Yu / Kedrick McKissock / Antonette Bennett / Judit Penzes / Bridget Lins-Austin / Qian Yu / Paul Chipman / Nilakshee Bhattacharya / Duncan Sousa / David Strugatsky / Peter Tijssen / Robert McKenna / Mavis Agbandje-McKenna /
Abstract: Adeno-associated viruses (AAVs) are being developed as clinical gene therapy vectors. One issue undermining their broad use in the clinical setting is the high prevalence of circulating antibodies in ...Adeno-associated viruses (AAVs) are being developed as clinical gene therapy vectors. One issue undermining their broad use in the clinical setting is the high prevalence of circulating antibodies in the general population capable of neutralizing AAV vectors. Hence, there is a need for AAV vectors that can evade the preexisting immune response. One possible source of human naive vectors are AAVs that do not disseminate in the primate population, and one such example is serpentine AAV (SAAV). This study characterizes the structural and biophysical properties of the SAAV capsid and its receptor interactions and antigenicity. Single particle cryo-electron microscopy (cryo-EM) and thermal stability studies were conducted to characterize the SAAV capsid structure at pH 7.4, 6.0, 5.5, and 4.0, conditions experienced during cellular trafficking. Cell binding assays using Chinese hamster ovary (CHO) cell lines identified terminal sialic acid as the primary attachment receptor for SAAV similar to AAV1, 4, 5, and 6. The binding site of sialic acid to the SAAV capsid was mapped near the 2-fold axis toward the 2/5-fold wall, in a different location than AAV1, 4, 5, and 6. Towards determining the SAAV capsid antigenicity native immunodot blots showed that SAAV evades AAV serotype-specific mouse monoclonal antibodies. However, despite its reptilian origin, it was recognized by ~25% of 50 human sera tested, likely due to the presence of cross-reactive antibodies. These findings will inform future gene delivery applications using SAAV-based vectors and further aid the structural characterization and annotation of the repertoire of available AAV capsids. AAVs are widely studied therapeutic gene delivery vectors. However, preexisting antibodies and their detrimental effect on therapeutic efficacy are a primary challenge encountered during clinical trials. In order to circumvent preexisting neutralizing antibodies targeting mammalian AAV capsids, serpentine AAV (SAAV) was evaluated as a potential alternative to existing mammalian therapeutic vectors. The SAAV capsid was found to be thermostable at a wide range of environmental pH conditions, and its structure showed conservation of the core capsid topology but displays high structural variability on the surface. At the same time, it binds to a common receptor, sialic acid, that is also utilized by other AAVs already being utilized in gene therapy trials. Contrary to the initial hypothesis, SAAV capsids were recognized by one in four human sera tested, pointing to conserved amino acids around the 5-fold region as epitopes for cross-reacting antibodies.
History
DepositionMar 10, 2022-
Header (metadata) releaseApr 13, 2022-
Map releaseApr 13, 2022-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26390.png

Downloads & links

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Map

FileDownload / File: emd_26390.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)X (Row.)Y (Col.)
1.24 Å/pix.
x 400 pix.
= 496. Å		1.24 Å/pix.
x 400 pix.
= 496. Å		1.24 Å/pix.
x 400 pix.
= 496. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.24 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0
Minimum - Maximum-9.651579 - 21.969363999999999
Average (Standard dev.)-0.000000003567578 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-200-200-200
Dimensions400400400
Spacing400400400
CellA=B=C: 496.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_26390_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26390_half_map_2.map
Projections & Slices
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Sample components

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Entire : Snake adeno-associated virus

EntireName: Snake adeno-associated virus
Components
  • Virus: Snake adeno-associated virus
    • Protein or peptide: Capsid protein

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Supramolecule #1: Snake adeno-associated virus

SupramoleculeName: Snake adeno-associated virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 252602 / Sci species name: Snake adeno-associated virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes

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Macromolecule #1: Capsid protein

MacromoleculeName: Capsid protein / type: protein_or_peptide / ID: 1 / Number of copies: 60 / Enantiomer: LEVO
Source (natural)Organism: Snake adeno-associated virus
Molecular weightTheoretical: 57.918895 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: AEGVGNSSGD WHCDTKWMGD HVITKSTRTW VLPTYGNHLY GPINFDGTTG SGANAAYAGY KTPWGYFDFN RFHCHFSPRD WQRLINNHT GIRPKGLKIK VFNVQVKEVT TQDSTKTIAN NLTSTVQIFA DENYDLPYVL GSATQGTFPP FPNDVFMLPQ Y AYCTLQGN ...String:
AEGVGNSSGD WHCDTKWMGD HVITKSTRTW VLPTYGNHLY GPINFDGTTG SGANAAYAGY KTPWGYFDFN RFHCHFSPRD WQRLINNHT GIRPKGLKIK VFNVQVKEVT TQDSTKTIAN NLTSTVQIFA DENYDLPYVL GSATQGTFPP FPNDVFMLPQ Y AYCTLQGN SGKFVDRSAF YCLEYFPSQM LRTGNNFEFQ FKFEEVPFHS GWAQSQSLDR LMNPLLDQYL IGDYGTDASG NL IYHRAGP NDLNEFYKNW APAPYECIQN INSSDNTKNA NSINGSNSTN KWGLQGRQAW DAPGFVQAST YEGAAAGQSL LNG VLTFDK SSATTSSPAA TAVNRTIEDE IQGTNNFGNA RNNIVAINQQ TKGTNPTTGS TSQFETMPGM VWSNRDIYLQ GPIW AKIPN TDGHFHPSPR MGGFGLKHPP PMILIKNTPV PADPPTTFNP MPQTSFITEY STGQVTVEML WEVQKESSKR WNPEV QFTS NFGTSDPAVD GIPFGINNLG TYVESRPIGT RYISKHL

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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Average electron dose: 62.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 73613
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: PROJECTION MATCHING

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