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- EMDB-24494: Cryo-EM Structure of Adeno-Associated Virus Serotype 9 with Engin... -

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Basic information

Entry
Database: EMDB / ID: EMD-24494
TitleCryo-EM Structure of Adeno-Associated Virus Serotype 9 with Engineered Peptide Domain PHP.B (AAV9-PHP.B)
Map dataAdeno-Associated Virus Serotype 9 with Engineered Peptide Domain PHP.B (AAV9-PHP.B)
Sample
  • Virus: Adeno-associated virus 9
    • Protein or peptide: Capsid protein VP1
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 9
Methodsingle particle reconstruction / cryo EM / Resolution: 2.27 Å
AuthorsFluck EC / Pumroy RA / Moiseenkova-Bell VY
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM103899 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM129357 United States
CitationJournal: J Virol / Year: 2021
Title: Context-Specific Function of the Engineered Peptide Domain of PHP.B.
Authors: R Alexander Martino / Edwin C Fluck / Jacqueline Murphy / Qiang Wang / Henry Hoff / Ruth A Pumroy / Claudia Y Lee / Joshua J Sims / Soumitra Roy / Vera Y Moiseenkova-Bell / James M Wilson /
Abstract: One approach to improve the utility of adeno-associated virus (AAV)-based gene therapy is to engineer the AAV capsid to (i) overcome poor transport through tissue barriers and (ii) redirect the ...One approach to improve the utility of adeno-associated virus (AAV)-based gene therapy is to engineer the AAV capsid to (i) overcome poor transport through tissue barriers and (ii) redirect the broadly tropic AAV to disease-relevant cell types. Peptide- or protein-domain insertions into AAV surface loops can achieve both engineering goals by introducing a new interaction surface on the AAV capsid. However, we understand little about the impact of insertions on capsid structure and the extent to which engineered inserts depend on a specific capsid context to function. Here, we examine insert-capsid interactions for the engineered variant AAV9-PHP.B. The 7-amino-acid peptide insert in AAV9-PHP.B facilitates transport across the murine blood-brain barrier via binding to the receptor Ly6a. When transferred to AAV1, the engineered peptide does not bind Ly6a. Comparative structural analysis of AAV1-PHP.B and AAV9-PHP.B revealed that the inserted 7-amino-acid loop is highly flexible and has remarkably little impact on the surrounding capsid conformation. Our work demonstrates that Ly6a binding requires interactions with both the PHP.B peptide and specific residues from the AAV9 HVR VIII region. An AAV1-based vector that incorporates a larger region of AAV9-PHP.B-including the 7-amino-acid loop and adjacent HVR VIII amino acids-can bind to Ly6a and localize to brain tissue. However, unlike AAV9-PHP.B, this AAV1-based vector does not penetrate the blood-brain barrier. Here we discuss the implications for AAV capsid engineering and the transfer of engineered activities between serotypes. Targeting AAV vectors to specific cellular receptors is a promising strategy for enhancing expression in target cells or tissues while reducing off-target transgene expression. The AAV9-PHP.B/Ly6a interaction provides a model system with a robust biological readout that can be interrogated to better understand the biology of AAV vectors' interactions with target receptors. In this work, we analyzed the sequence and structural features required to successfully transfer the Ly6a receptor-binding epitope from AAV9-PHP.B to another capsid of clinical interest, AAV1. We found that AAV1- and AAV9-based vectors targeted to the same receptor exhibited different brain-transduction profiles. Our work suggests that, in addition to attachment-receptor binding, the capsid context in which this binding occurs is important for a vector's performance.
History
DepositionJul 22, 2021-
Header (metadata) releaseAug 4, 2021-
Map releaseAug 4, 2021-
UpdateOct 6, 2021-
Current statusOct 6, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
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  • Surface view colored by radius
  • Surface level: 0.04
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  • Surface view with fitted model
  • Atomic models: PDB-7rk8
  • Surface level: 0.04
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7rk8
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24494.png

Downloads & links

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Map

FileDownload / File: emd_24494.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAdeno-Associated Virus Serotype 9 with Engineered Peptide Domain PHP.B (AAV9-PHP.B)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.115297794 - 0.21087085
Average (Standard dev.)3.1664837e-05 (±0.010908328)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 457.91998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z432432432
origin x/y/z0.0000.0000.000
length x/y/z457.920457.920457.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS432432432
D min/max/mean-0.1150.2110.000

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Supplemental data

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Sample components

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Entire : Adeno-associated virus 9

EntireName: Adeno-associated virus 9
Components
  • Virus: Adeno-associated virus 9
    • Protein or peptide: Capsid protein VP1

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Supramolecule #1: Adeno-associated virus 9

SupramoleculeName: Adeno-associated virus 9 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: AAV9-PHP.B trans plasmid with cis plasmid encoding the CB7-EGFP reporter gene to produce vectors in HEK293 cells via triple transfection.
NCBI-ID: 235455 / Sci species name: Adeno-associated virus 9 / Virus type: SATELLITE / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No
Host systemOrganism: Homo sapiens (human) / Recombinant strain: 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 9
Molecular weightTheoretical: 82.215672 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 HQSAQTLAVP FKAQAQTGWV QNQGILPGMV WQDRDVYLQG PIWAKIPHTD GNFHPSP LM GGFGMKHPPP QILIKNTPVP ADPPTAFNKD KLNSFITQYS TGQVSVEIEW ELQKENSKRW NPEIQYTSNY YKSNNVEF A VNTEGVYSEP RPIGTRYLTR NL

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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
Component:
ConcentrationFormulaName
137.0 mMNaClSodium chloridesodium chloride
2.7 mMKClpotassium chloride
10.0 mMNa2HPO4sodium phosphate dibasic
1.8 mMKH2PO4potassium phosphate monobasic

Details: Supplemented with 0.001% Pluronic F68
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Support film - Film thickness: 3.0 nm
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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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
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 1380 / Average electron dose: 38.1 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 89358
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER / Details: RELION Ab-initio model
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.27 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 42570
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-7rk8:
Cryo-EM Structure of Adeno-Associated Virus Serotype 9 with Engineered Peptide Domain PHP.B (AAV9-PHP.B)

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