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- EMDB-41918: 3-fold symmetry face of adeno-associated virus-9 and human Interl... -

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Basic information

Entry
Database: EMDB / ID: EMD-41918
Title3-fold symmetry face of adeno-associated virus-9 and human Interleukin 3 complex
Map data
Sample
  • Complex: Adeno-associated virus-9 and human interleukin 3 complex
    • Protein or peptide: Adeno-associated virus-9
    • Protein or peptide: Human Interleukin 3 triple-tagged with Fc-Myc-8xHis
KeywordsAdeno-associated virus / AAV9 / interleukin 3 / cryo-electron tomography / VIRUS LIKE PARTICLE
Biological speciesAdeno-associated virus 9 / Homo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 20.0 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 12, 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_41918.png

Downloads & links

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Map

FileDownload / File: emd_41918.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3 Å/pix.
x 72 pix.
= 216. Å		3 Å/pix.
x 72 pix.
= 216. Å		3 Å/pix.
x 72 pix.
= 216. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.065
Minimum - Maximum-0.46251282 - 0.7550747
Average (Standard dev.)0.051129855 (±0.17346963)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA=B=C: 216.0 Å
α=β=γ: 90.0 °

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Supplemental data

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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
    • Protein or peptide: Adeno-associated virus-9
    • Protein or peptide: Human Interleukin 3 triple-tagged with Fc-Myc-8xHis

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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: all
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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Macromolecule #1: Adeno-associated virus-9

MacromoleculeName: Adeno-associated virus-9 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Adeno-associated virus 9
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR ...String:
MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE SVPDPQPIGE PPAAPSGVGS LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY QLPYVLGSAH EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENV PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT INGSGQNQQT LKFSVAGPSN MAVQGRNYIP GPSYRQQRVS TTVTQNNNSE FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS LIFGKQGTGR DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VEFAVNTEGV YSEPRPIGTR YLTRNL

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Macromolecule #2: Human Interleukin 3 triple-tagged with Fc-Myc-8xHis

MacromoleculeName: Human Interleukin 3 triple-tagged with Fc-Myc-8xHis / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSRLPVLLLL QLLVRPGLQA PMTQTTPLKT SWVNCSNMID EIITHLKQPP LPLLDFNNLN GEDQDILMEN NLRRPNLEAF NRAVKSLQNA SAIESILKNL LPCLPLATAA PTRHPIHIKD GDWNEFRRKL TFYLKTLENA QAQQTTLSLA IFGAPENLYF QGGGDKTHTC ...String:
MSRLPVLLLL QLLVRPGLQA PMTQTTPLKT SWVNCSNMID EIITHLKQPP LPLLDFNNLN GEDQDILMEN NLRRPNLEAF NRAVKSLQNA SAIESILKNL LPCLPLATAA PTRHPIHIKD GDWNEFRRKL TFYLKTLENA QAQQTTLSLA IFGAPENLYF QGGGDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVAV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALAAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG GGSGGSEQKL ISEEDLGGSH HHHHHHH

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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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 10mA
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: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: RELION (ver. 3.1.3) / Number subtomograms used: 108474
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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