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- EMDB-24003: Structure of the adeno-associated virus 9 capsid at pH pH 5.5 in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-24003
TitleStructure of the adeno-associated virus 9 capsid at pH pH 5.5 in complex with terminal galactose
Map data
Sample
  • Virus: Adeno-associated virus 9
    • Protein or peptide: Capsid protein VP1
  • Ligand: beta-D-galactopyranose
Keywordsadeno-associated virus / pH / phospholipase-A2 / capsid / gene therapy / AAV9 / dependoparvovirus / intracellular trafficking / endosome / VIRUS LIKE PARTICLE / galactose / glycan attachment
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: 2.68 Å
AuthorsPenzes JJ / Chipman P
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 14, 2021-
Header (metadata) releaseJun 9, 2021-
Map releaseJun 9, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing 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-7mua
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7mua
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24003.png

Downloads & links

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Map

FileDownload / File: emd_24003.map.gz / Format: CCP4 / Size: 729 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
1.05 Å/pix.
x 576 pix.
= 603.648 Å		1.05 Å/pix.
x 576 pix.
= 603.648 Å		1.05 Å/pix.
x 576 pix.
= 603.648 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.048 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 2.0 / Movie #1: 2
Minimum - Maximum-14.540972999999999 - 31.63232
Average (Standard dev.)-0.000000000931133 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-288-288-288
Dimensions576576576
Spacing576576576
CellA=B=C: 603.648 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0481.0481.048
M x/y/z576576576
origin x/y/z0.0000.0000.000
length x/y/z603.648603.648603.648
α/β/γ90.00090.00090.000
start NX/NY/NZ-288-288-288
NX/NY/NZ576576576
MAP C/R/S213
start NC/NR/NS-288-288-288
NC/NR/NS576576576
D min/max/mean-14.54131.632-0.000

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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
  • Ligand: beta-D-galactopyranose

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Supramolecule #1: Adeno-associated virus 9

SupramoleculeName: Adeno-associated virus 9 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1 / 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)
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

UniProtKB: Capsid protein VP1

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Macromolecule #2: beta-D-galactopyranose

MacromoleculeName: beta-D-galactopyranose / type: ligand / ID: 2 / Number of copies: 60 / Formula: GAL
Molecular weightTheoretical: 180.156 Da
Chemical component information

ChemComp-GAL:
beta-D-galactopyranose

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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: 5.5
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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 1445 / Average electron dose: 63.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: OTHER / Cs: 2.7 mm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 134697
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio random model
Final reconstructionNumber classes used: 15 / Applied symmetry - Point group: I (icosahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0) / Number images used: 110690
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final 3D classificationNumber classes: 35 / Software - Name: cisTEM (ver. 1.0.0)

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Atomic model buiding 1

Initial modelPDB ID:

3ux1


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7mua:
Structure of the adeno-associated virus 9 capsid at pH pH 5.5 in complex with terminal galactose

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