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

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Basic information

Entry
Database: PDB / ID: 7ud4
TitleCryo-EM structure of AAV-PHP.eB
ComponentsCapsid protein VP1
KeywordsVIRUS / Gene therapy / AAV / capsid / blood-brain barrier / directed evolution
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
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.24 Å
AuthorsJang, S. / Shen, H.K. / Ding, X. / Miles, T.F. / Gradinaru, V.
Funding support United States, 1items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Capsid protein VP1


Theoretical massNumber of molelcules
Total (without water)82,1891
Polymers82,1891
Non-polymers00
Water00
1
A: Capsid protein VP1
x 60


  • defined by software
  • Evidence: electron microscopy
  • 4.93 MDa, 60 polymers
Theoretical massNumber of molelcules
Total (without water)4,931,31760
Polymers4,931,31760
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
MethodUCSF CHIMERA

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Components

#1: Protein Capsid protein VP1


Mass: 82188.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus / Gene: cap / Production host: Homo sapiens (human) / References: UniProt: Q6JC40

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Adeno-associated virus / Type: VIRUS / Details: AAV-PHP.eB: Engineered AAV from AAV9. / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Adeno-associated virus
Source (recombinant)Organism: Homo sapiens (human) / Cell: AAVpro293T
Details of virusEmpty: NO / Enveloped: NO / Isolate: SEROTYPE / Type: VIRUS-LIKE PARTICLE
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 4000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameCategory
2cryoSPARCimage acquisition
7Cootmodel fitting
9PHENIXmodel refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50195 / Symmetry type: POINT
Atomic model buildingPDB-ID: 3UX1

3ux1


Accession code: 3UX1 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0044289
ELECTRON MICROSCOPYf_angle_d0.4755842
ELECTRON MICROSCOPYf_dihedral_angle_d4.697567
ELECTRON MICROSCOPYf_chiral_restr0.042597
ELECTRON MICROSCOPYf_plane_restr0.004778

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