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- EMDB-23986: Structure of the adeno-associated virus 9 capsid at pH 6.0 -

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Basic information

Entry
Database: EMDB / ID: EMD-23986
TitleStructure of the adeno-associated virus 9 capsid at pH 6.0
Map dataAAV9 capsid at pH 6.0
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: 2.67 Å
AuthorsPenzes JJ / Chipman P / Bhattacharya N / Zeher A / Huang R / McKenna R / Agbandje-McKenna M
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM109524 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM082946 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10 OD018142 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10 RR025080 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM116788 United States
CitationJournal: J Virol / Year: 2021
Title: Adeno-associated Virus 9 Structural Rearrangements Induced by Endosomal Trafficking pH and Glycan Attachment.
Authors: Judit J Penzes / Paul Chipman / Nilakshee Bhattacharya / Allison Zeher / Rick Huang / Robert McKenna / Mavis Agbandje-McKenna /
Abstract: Adeno-associated viruses (AAVs) are small nonenveloped single-stranded DNA (ssDNA) viruses that are currently being developed as gene therapy biologics. After cell entry, AAVs traffic to the nucleus ...Adeno-associated viruses (AAVs) are small nonenveloped single-stranded DNA (ssDNA) viruses that are currently being developed as gene therapy biologics. After cell entry, AAVs traffic to the nucleus using the endo-lysosomal pathway. The subsequent decrease in pH triggers conformational changes to the capsid that enable the externalization of the capsid protein (VP) N termini, including the unique domain of the minor capsid protein VP1 (VP1u), which permits the phospholipase activity required for the capsid lysosomal egress. Here, we report the AAV9 capsid structure, determined at the endosomal pHs (7.4, 6.0, 5.5, and 4.0), and terminal galactose-bound AAV9 capsids at pHs 7.4 and 5.5 using cryo-electron microscopy and three-dimensional image reconstruction. Taken together, these studies provide insight into AAV9 capsid conformational changes at the 5-fold pore during endosomal trafficking, in both the presence and absence of its cellular glycan receptor. We visualized, for the first time, that acidification induces the externalization of the VP3 and possibly VP2 N termini, presumably in prelude to the externalization of VP1u at pH 4.0, which is essential for lysosomal membrane disruption. In addition, the structural study of AAV9-galactose interactions demonstrates that AAV9 remains attached to its glycan receptor at the late endosome pH 5.5. This interaction significantly alters the conformational stability of the variable region I of the VPs, as well as the dynamics associated with VP N terminus externalization. There are 13 distinct Adeno-associated virus (AAV) serotypes that are structurally homologous and whose capsid proteins (VP1 to -3) are similar in amino acid sequence. However, AAV9 is one of the most commonly studied and is used as a gene therapy vector. This is partly because AAV9 is capable of crossing the blood-brain barrier and readily transduces a wide array of tissues, including the central nervous system. In this study, we provide AAV9 capsid structural insight during intracellular trafficking. Although the AAV capsid has been shown to externalize the N termini of its VPs, to enzymatically disrupt the lysosome membrane at low pH, there was no structural evidence to confirm this. By utilizing AAV9 as our model, we provide the first structural evidence that the externalization process occurs at the protein interface at the icosahedral 5-fold symmetry axis and can be triggered by lowering the pH.
History
DepositionMay 13, 2021-
Header (metadata) releaseJul 21, 2021-
Map releaseJul 21, 2021-
UpdateSep 22, 2021-
Current statusSep 22, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mtg
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7mtg
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23986.png

Downloads & links

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Map

FileDownload / File: emd_23986.map.gz / Format: CCP4 / Size: 327.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAAV9 capsid at pH 6.0
Voxel sizeX=Y=Z: 1.087 Å
Density
Contour LevelBy EMDB: 2.0 / Movie #1: 2
Minimum - Maximum-14.99657 - 25.952127
Average (Standard dev.)-3.126984e-09 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-220-220-220
Dimensions441441441
Spacing441441441
CellA=B=C: 479.367 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0871.0871.087
M x/y/z441441441
origin x/y/z0.0000.0000.000
length x/y/z479.367479.367479.367
α/β/γ90.00090.00090.000
start NX/NY/NZ-220-220-220
NX/NY/NZ441441441
MAP C/R/S213
start NC/NR/NS-220-220-220
NC/NR/NS441441441
D min/max/mean-14.99725.952-0.000

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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 / NCBI-ID: 235455 / Sci species name: Adeno-associated virus 9 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human)
Host systemOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9 / Recombinant plasmid: pFastBac1-HM
Virus shellShell ID: 1 / Name: AAV9 virus like particle / Diameter: 240.0 Å / T number (triangulation number): 1

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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: 58.474414 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DGVGSSSGNW HCDSQWLGDR VITTSTRTWA LPTYNNHLYK QISNSTSGGS SNDNAYFGYS TPWGYFDFNR FHCHFSPRDW QRLINNNWG FRPKRLNFKL FNIQVKEVTD NNGVKTIANN LTSTVQVFTD SDYQLPYVLG SAHEGCLPPF PADVFMIPQY G YLTLNDGS ...String:
DGVGSSSGNW HCDSQWLGDR VITTSTRTWA LPTYNNHLYK QISNSTSGGS SNDNAYFGYS TPWGYFDFNR FHCHFSPRDW QRLINNNWG FRPKRLNFKL FNIQVKEVTD NNGVKTIANN LTSTVQVFTD SDYQLPYVLG SAHEGCLPPF PADVFMIPQY G YLTLNDGS QAVGRSSFYC LEYFPSQMLR TGNNFQFSYE FENVPFHSSY AHSQSLDRLM NPLIDQYLYY LSKTINGSGQ NQ QTLKFSV AGPSNMAVQG RNYIPGPSYR QQRVSTTVTQ NNNSEFAWPG ASSWALNGRN SLMNPGPAMA SHKEGEDRFF PLS GSLIFG KQGTGRDNVD ADKVMITNEE EIKTTNPVAT ESYGQVATNH QSAQAQAQTG WVQNQGILPG MVWQDRDVYL QGPI WAKIP HTDGNFHPSP LMGGFGMKHP PPQILIKNTP VPADPPTAFN KDKLNSFITQ YSTGQVSVEI EWELQKENSK RWNPE IQYT SNYYKSNNVE FAVNTEGVYS EPRPIGTRYL TRNL

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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
Details: 1x Universal buffer of 20 mM HEPES, 20 mM MES, 20 mM sodium acetate, 0.15 M NaCl, 3.7 mM CaCl2
GridModel: Quantifoil / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: OTHER / Cs: 2.7 mm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Auto3DEM (ver. 5.04.2) / Software - details: Autopp3X subroutine
Startup modelType of model: INSILICO MODEL
In silico model: Ab initio random model based on 100 boxed particles
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.67 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Auto3DEM (ver. 5.02.4) / Number images used: 107405

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Atomic model buiding 1

Initial modelPDB ID:

3ux1


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7mtg:
Structure of the adeno-associated virus 9 capsid at pH 6.0

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