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- EMDB-42063: I1 symmetry applied adeno-associated virus-9 with human Interleukin 3 -

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Basic information

Entry
Database: EMDB / ID: EMD-42063
TitleI1 symmetry applied adeno-associated virus-9 with human Interleukin 3
Map data
Sample
  • Complex: Adeno-associated virus-9 and human interleukin 3 complex
KeywordsAdeno-associated virus / AAV9 / interleukin 3 / cryo-electron tomography / VIRUS LIKE PARTICLE
Biological speciesAdeno-associated virus 9
Methodsubtomogram averaging / cryo EM / Resolution: 11.0 Å
AuthorsBrittain T / Jang S / Shay TF / Chen X / Gradinaru V
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)DP1.NS111369 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)UF1.MH128336 United States
CitationJournal: Nat Commun / Year: 2024
Title: Human cell surface-AAV interactomes identify LRP6 as blood-brain barrier transcytosis receptor and immune cytokine IL3 as AAV9 binder.
Authors: Timothy F Shay / Seongmin Jang / Tyler J Brittain / Xinhong Chen / Beth Walker / Claire Tebbutt / Yujie Fan / Damien A Wolfe / Cynthia M Arokiaraj / Erin E Sullivan / Xiaozhe Ding / Ting-Yu ...Authors: Timothy F Shay / Seongmin Jang / Tyler J Brittain / Xinhong Chen / Beth Walker / Claire Tebbutt / Yujie Fan / Damien A Wolfe / Cynthia M Arokiaraj / Erin E Sullivan / Xiaozhe Ding / Ting-Yu Wang / Yaping Lei / Miguel R Chuapoco / Tsui-Fen Chou / Viviana Gradinaru /
Abstract: Adeno-associated viruses (AAVs) are foundational gene delivery tools for basic science and clinical therapeutics. However, lack of mechanistic insight, especially for engineered vectors created by ...Adeno-associated viruses (AAVs) are foundational gene delivery tools for basic science and clinical therapeutics. However, lack of mechanistic insight, especially for engineered vectors created by directed evolution, can hamper their application. Here, we adapt an unbiased human cell microarray platform to determine the extracellular and cell surface interactomes of natural and engineered AAVs. We identify a naturally-evolved and serotype-specific interaction between the AAV9 capsid and human interleukin 3 (IL3), with possible roles in host immune modulation, as well as lab-evolved low-density lipoprotein receptor-related protein 6 (LRP6) interactions specific to engineered capsids with enhanced blood-brain barrier crossing in non-human primates after intravenous administration. The unbiased cell microarray screening approach also allows us to identify off-target tissue binding interactions of engineered brain-enriched AAV capsids that may inform vectors' peripheral organ tropism and side effects. Our cryo-electron tomography and AlphaFold modeling of capsid-interactor complexes reveal LRP6 and IL3 binding sites. These results allow confident application of engineered AAVs in diverse organisms and unlock future target-informed engineering of improved viral and non-viral vectors for non-invasive therapeutic delivery to the brain.
History
DepositionSep 20, 2023-
Header (metadata) releaseSep 18, 2024-
Map releaseSep 18, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42063.png

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Map

FileDownload / File: emd_42063.map.gz / Format: CCP4 / Size: 8.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
3 Å/pix.
x 132 pix.
= 396. Å		3 Å/pix.
x 132 pix.
= 396. Å		3 Å/pix.
x 132 pix.
= 396. Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 3 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.23
Minimum - Maximum-0.013393884 - 0.41915232
Average (Standard dev.)0.05620585 (±0.10913996)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions132132132
Spacing132132132
CellA=B=C: 396.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_42063_half_map_1.map
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Half map: #2

Fileemd_42063_half_map_2.map
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Sample components

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Entire : Adeno-associated virus-9 and human interleukin 3 complex

EntireName: Adeno-associated virus-9 and human interleukin 3 complex
Components
  • Complex: Adeno-associated virus-9 and human interleukin 3 complex

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Supramolecule #1: Adeno-associated virus-9 and human interleukin 3 complex

SupramoleculeName: Adeno-associated virus-9 and human interleukin 3 complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: A complex of adeno-associated virus-9 and human Interleukin 3. Both proteins were purified from a human cell-based expression system.
Source (natural)Organism: Adeno-associated virus 9
Molecular weightTheoretical: 46.8 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6 / Component:
ConcentrationName
20.0 mMPBS
0.001 %Pluronic-F68
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number real images: 1 / Average exposure time: 0.474 sec. / Average electron dose: 1.5 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: 3.726 µm / Nominal defocus min: 2.813 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.3) / Number subtomograms used: 2661
ExtractionNumber tomograms: 46 / Number images used: 2661 / Method: particles picked manually
Software:
Namedetails
Dynamo (ver. 1.1532)Particle picking
Warp (ver. 1.0.9)Sub Volume extraction
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3) / Details: Relion Maximum Likelihood
FSC plot (resolution estimation)

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