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- EMDB-24495: Cryo-EM Structure of Adeno-Associated Virus Serotype 1 with Engin... -

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Basic information

Entry
Database: EMDB / ID: EMD-24495
TitleCryo-EM Structure of Adeno-Associated Virus Serotype 1 with Engineered Peptide Domain PHP.B (AAV1-PHP.B)
Map dataAdeno-Associated Virus Serotype 1 with Engineered Peptide Domain PHP.B (AAV1-PHP.B)
Sample
  • Virus: Adeno-associated virus - 1
    • Protein or peptide: Capsid protein
KeywordsGene Therapy / Adeno-associated virus / AAV / 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
Function and homology information
Biological speciesAdeno-associated virus - 1
Methodsingle particle reconstruction / cryo EM / Resolution: 2.32 Å
AuthorsFluck EC / Pumroy RA
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-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.025
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  • Surface view with fitted model
  • Atomic models: PDB-7rk9
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7rk9
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24495.png

Downloads & links

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Map

FileDownload / File: emd_24495.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAdeno-Associated Virus Serotype 1 with Engineered Peptide Domain PHP.B (AAV1-PHP.B)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 480 pix.
= 417.6 Å		0.87 Å/pix.
x 480 pix.
= 417.6 Å		0.87 Å/pix.
x 480 pix.
= 417.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.87 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.075143255 - 0.120654285
Average (Standard dev.)0.00018174619 (±0.0067386134)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 417.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.870.870.87
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z417.600417.600417.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.0750.1210.000

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Supplemental data

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Sample components

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Entire : Adeno-associated virus - 1

EntireName: Adeno-associated virus - 1
Components
  • Virus: Adeno-associated virus - 1
    • Protein or peptide: Capsid protein

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Supramolecule #1: Adeno-associated virus - 1

SupramoleculeName: Adeno-associated virus - 1 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: AAV1-PHP.B trans plasmids with cis plasmids encoding the CB7-EGFP reporter gene to produce vectors in HEK293 cells via triple transfection.
NCBI-ID: 85106 / Sci species name: Adeno-associated virus - 1 / Virus type: SATELLITE / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No

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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: Adeno-associated virus - 1
Molecular weightTheoretical: 82.212859 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNP YLRYNHADAE FQERLQEDTS FGGNLGRAVF QAKKRVLEPL GLVEEGAKTA PGKKRPVEQS PQEPDSSSGI G KTGQQPAK ...String:
MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNP YLRYNHADAE FQERLQEDTS FGGNLGRAVF QAKKRVLEPL GLVEEGAKTA PGKKRPVEQS PQEPDSSSGI G KTGQQPAK KRLNFGQTGD SESVPDPQPL GEPPATPAAV GPTTMASGGG APMADNNEGA DGVGNASGNW HCDSTWLGDR VI TTSTRTW ALPTYNNHLY KQISSASTGA SNDNHYFGYS TPWGYFDFNR FHCHFSPRDW QRLINNNWGF RPKRLNFKLF NIQ VKEVTT NDGVTTIANN LTSTVQVFSD SEYQLPYVLG SAHQGCLPPF PADVFMIPQY GYLTLNNGSQ AVGRSSFYCL EYFP SQMLR TGNNFTFSYT FEEVPFHSSY AHSQSLDRLM NPLIDQYLYY LNRTQNQSGS AQNKDLLFSR GSPAGMSVQP KNWLP GPCY RQQRVSKTKT DNNNSNFTWT GASKYNLNGR ESIINPGTAM ASHKDDEDKF FPMSGVMIFG KESAGASNTA LDNVMI TDE EEIKATNPVA TERFGTVAVN FQSSSTLAVP FKTDPATGDV HAMGALPGMV WQDRDVYLQG PIWAKIPHTD GHFHPSP LM GGFGLKNPPP QILIKNTPVP ANPPAEFSAT KFASFITQYS TGQVSVEIEW ELQKENSKRW NPEVQYTSNY AKSANVDF T VDNNGLYTEP RPIGTRYLTR PL

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
Component:
ConcentrationFormulaName
137.0 mMNaClsodium chloride
2.7 mMKClpotassium chloride
10.0 mMNa2HPO4sodium phosphate dibasic
1.8 mMKH2P04potassium 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 1950 / Average electron dose: 37.5 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 151543
Startup modelType of model: OTHER / Details: RELION Ab-initio model
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.32 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 59346
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-7rk9:
Cryo-EM Structure of Adeno-Associated Virus Serotype 1 with Engineered Peptide Domain PHP.B (AAV1-PHP.B)

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