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- EMDB-26453: Cryo-EM structure of AAV-PHP.eB, I1 symmetry applied -

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Basic information

Entry
Database: EMDB / ID: EMD-26453
TitleCryo-EM structure of AAV-PHP.eB, I1 symmetry applied
Map dataCryo-EM structure of AAV-PHP.eB
Sample
  • Virus: Adeno-associated virus
    • Protein or peptide: Capsid protein VP1
KeywordsGene therapy / AAV / capsid / blood-brain barrier / directed evolution / VIRUS
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: 2.24 Å
AuthorsJang S / Shen HK / Ding X / Miles TF / Gradinaru V
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)DP1.NS111369 United States
CitationJournal: Mol Ther Methods Clin Dev / Year: 2022
Title: Structural basis of receptor usage by the engineered capsid AAV-PHP.eB.
Authors: Seongmin Jang / Hao K Shen / Xiaozhe Ding / Timothy F Miles / Viviana Gradinaru /
Abstract: Adeno-associated virus serotype 9 (AAV9) is a promising gene therapy vector for treating neurodegenerative diseases due to its ability to penetrate the blood-brain barrier. PHP.eB was engineered ...Adeno-associated virus serotype 9 (AAV9) is a promising gene therapy vector for treating neurodegenerative diseases due to its ability to penetrate the blood-brain barrier. PHP.eB was engineered from AAV9 by insertion of a 7-amino acid peptide and point mutation of neighboring residues, thereby enhancing potency in the central nervous system. Here, we report a 2.24-Å resolution cryo-electron microscopy structure of PHP.eB, revealing conformational differences from other 7-mer insertion capsid variants. In PHP.eB, the 7-mer loop adopts a bent conformation, mediated by an interaction between engineered lysine and aspartate residues. Further, we identify PKD2 as the main AAV receptor (AAVR) domain recognizing both AAV9 and PHP.eB and find that the PHP.eB 7-mer partially destabilizes this interaction. Analysis of previously reported AAV structures together with our pull-down data demonstrate that the 7-mer topology determined by the lysine-aspartate interaction dictates AAVR binding strength. Our results suggest that PHP.eB's altered tropism may arise from both an additional interaction with LY6A and weakening of its AAVR interaction. Changing the insertion length, but not sequence, modifies PKD2 binding affinity, suggesting that a steric clash impedes AAVR binding. This research suggests improved library designs for future AAV selections to identify non-LY6A-dependent vectors and modulate AAVR interaction strength.
History
DepositionMar 18, 2022-
Header (metadata) releaseSep 21, 2022-
Map releaseSep 21, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26453.png

Downloads & links

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Map

FileDownload / File: emd_26453.map.gz / Format: CCP4 / Size: 634.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of AAV-PHP.eB
Voxel sizeX=Y=Z: 0.869 Å
Density
Contour LevelBy AUTHOR: 0.471
Minimum - Maximum-0.7232382 - 2.0619142
Average (Standard dev.)0.0030179166 (±0.10800662)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions550550550
Spacing550550550
CellA=B=C: 477.95 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-map A

Fileemd_26453_half_map_1.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B

Fileemd_26453_half_map_2.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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 / Details: AAV-PHP.eB: Engineered AAV from AAV9. / NCBI-ID: 272636 / Sci species name: Adeno-associated virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No

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Macromolecule #1: Capsid protein VP1

MacromoleculeName: Capsid protein VP1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Adeno-associated virus
Molecular weightTheoretical: 82.188609 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNP YLKYNHADAE FQERLKEDTS FGGNLGRAVF QAKKRLLEPL GLVEEAAKTA PGKKRPVEQS PQEPDSSAGI G KSGAQPAK ...String:
MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNP YLKYNHADAE FQERLKEDTS FGGNLGRAVF QAKKRLLEPL GLVEEAAKTA PGKKRPVEQS PQEPDSSAGI G KSGAQPAK KRLNFGQTGD TESVPDPQPI GEPPAAPSGV GSLTMASGGG APVADNNEGA DGVGSSSGNW HCDSQWLGDR VI TTSTRTW ALPTYNNHLY KQISNSTSGG SSNDNAYFGY STPWGYFDFN RFHCHFSPRD WQRLINNNWG FRPKRLNFKL FNI QVKEVT DNNGVKTIAN NLTSTVQVFT DSDYQLPYVL GSAHEGCLPP FPADVFMIPQ YGYLTLNDGS QAVGRSSFYC LEYF PSQML RTGNNFQFSY EFENVPFHSS YAHSQSLDRL MNPLIDQYLY YLSKTINGSG QNQQTLKFSV AGPSNMAVQG RNYIP GPSY RQQRVSTTVT QNNNSEFAWP GASSWALNGR NSLMNPGPAM ASHKEGEDRF FPLSGSLIFG KQGTGRDNVD ADKVMI TNE EEIKTTNPVA TESYGQVATN HQSDGTLAVP FKAQAQTGWV QNQGILPGMV WQDRDVYLQG PIWAKIPHTD GNFHPSP LM GGFGMKHPPP QILIKNTPVP ADPPTAFNKD KLNSFITQYS TGQVSVEIEW ELQKENSKRW NPEIQYTSNY YKSNNVEF A VNTEGVYSEP RPIGTRYLTR NL

UniProtKB: Capsid protein VP1

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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 TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.24 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 50195
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: ANGULAR RECONSTITUTION

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Atomic model buiding 1

Initial modelPDB ID:

3ux1


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-7ud4:
Cryo-EM structure of AAV-PHP.eB

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