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- EMDB-46748: The Structure of AAV5 at 4 Degrees -

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Basic information

Entry
Database: EMDB / ID: EMD-46748
TitleThe Structure of AAV5 at 4 Degrees
Map data
Sample
  • Virus: adeno-associated virus 5
    • Protein or peptide: Capsid protein
    • DNA: DNA (5'-D(P*AP*A)-3')
Keywordstemperature / genome / vector / icosahedron / VIRUS
Function / homologyPhospholipase A2-like domain / Phospholipase A2-like domain / Parvovirus coat protein VP2 / Parvovirus coat protein VP1/VP2 / Parvovirus coat protein VP1/VP2 / Capsid/spike protein, ssDNA virus / T=1 icosahedral viral capsid / structural molecule activity / Capsid protein
Function and homology information
Biological speciesadeno-associated virus 5
Methodsingle particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsBennett AB / McKenna R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM082946 United States
CitationJournal: J Virol / Year: 2025
Title: Biophysical and structural insights into AAV genome ejection.
Authors: Keely Gliwa / Joshua Hull / Austin Kansol / Victoria Zembruski / Renuk Lakshmanan / Mario Mietzsch / Paul Chipman / Antonette Bennett / Robert McKenna /
Abstract: Recombinant adeno-associated virus (rAAV) is comprised of non-enveloped capsids that can package a therapeutic transgene and are currently being developed and utilized as gene therapy vectors. The ...Recombinant adeno-associated virus (rAAV) is comprised of non-enveloped capsids that can package a therapeutic transgene and are currently being developed and utilized as gene therapy vectors. The therapeutic efficiency of rAAV is dependent on successful cytoplasmic trafficking and transgene delivery to the nucleus. It is hypothesized that an increased understanding of the effects of the cellular environment and biophysical properties of the capsid as it traffics to the nucleus could provide insight to improve vector efficiency. The AAV capsid is exposed to increasing [H] during endo-lysosomal trafficking. Exposure to low pH facilitates the externalization of the viral protein 1 unique region (VP1u). This VP1u contains a phospholipase A2 domain required for endosomal escape and nuclear localization signals that facilitate nuclear targeting and entry. The viral genome is released either after total capsid disassembly or via a concerted DNA ejection mechanism in the nucleus. This study presents the characterization of genome ejection (GE) for two diverse serotypes, AAV2 and AAV5, using temperature. The temperature required to disassemble the virus capsid (T) is significantly higher than the temperature required to expose the transgene (T) for both serotypes. This was verified by quantitative PCR (qPCR) and transmission electron microscopy. Additionally, the absence of VP1/VP2 in the capsids and a decrease in pH increase the temperature of GE. Furthermore, cryo-electron microscopy structures of the AAV5 capsid pre- and post-GE reveal dynamics at the twofold, threefold, and fivefold regions of the capsid interior consistent with a concerted egress of the viral genome.IMPORTANCEThe development of recombinant adeno-associated virus (rAAV) capsids has grown rapidly in recent years, with five of the eight established therapeutics gaining approval in the past 2 years alone. Clinical progression with AAV2 and AAV5 represents a growing need to further characterize the molecular biology of these viruses. The goal of AAV-based gene therapy is to treat monogenic disorders with a vector-delivered transgene to provide wild-type protein function. A better understanding of the dynamics and conditions enabling transgene release may improve therapeutic efficiency. In addition to their clinical importance, AAV2 and 5 were chosen in this study for their diverse antigenic and biophysical properties compared to more closely related serotypes. Characterization of a shared genome ejection process may imply a conserved mechanism for all rAAV therapies.
History
DepositionAug 25, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateApr 16, 2025-
Current statusApr 16, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46748.png

Downloads & links

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Map

FileDownload / File: emd_46748.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 500 pix.
= 467.5 Å		0.94 Å/pix.
x 500 pix.
= 467.5 Å		0.94 Å/pix.
x 500 pix.
= 467.5 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.935 Å
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Histogram (log scale)
Contour LevelBy EMDB: 2.0
Minimum - Maximum-18.404845999999999 - 22.665665000000001
Average (Standard dev.)0.000005039154 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-250-250-250
Dimensions500500500
Spacing500500500
CellA=B=C: 467.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_46748_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_46748_half_map_2.map
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Sample components

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Entire : adeno-associated virus 5

EntireName: adeno-associated virus 5
Components
  • Virus: adeno-associated virus 5
    • Protein or peptide: Capsid protein
    • DNA: DNA (5'-D(P*AP*A)-3')

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Supramolecule #1: adeno-associated virus 5

SupramoleculeName: adeno-associated virus 5 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Baculovirus expression / NCBI-ID: 82300 / Sci species name: adeno-associated virus 5 / Virus type: VIRION / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No
Molecular weightTheoretical: 4 MDa

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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 5
Molecular weightTheoretical: 80.497414 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSFVDHPPDW LEEVGEGLRE FLGLEAGPPK PKPNQQHQDQ ARGLVLPGYN YLGPGNGLDR GEPVNRADEV AREHDISYNE QLEAGDNPY LKYNHADAEF QEKLADDTSF GGNLGKAVFQ AKKRVLEPFG LVEEGAKTAP TGKRIDDHFP KRKKARTEED S KPSTSSDA ...String:
MSFVDHPPDW LEEVGEGLRE FLGLEAGPPK PKPNQQHQDQ ARGLVLPGYN YLGPGNGLDR GEPVNRADEV AREHDISYNE QLEAGDNPY LKYNHADAEF QEKLADDTSF GGNLGKAVFQ AKKRVLEPFG LVEEGAKTAP TGKRIDDHFP KRKKARTEED S KPSTSSDA EAGPSGSQQL QIPAQPASSL GADTMSAGGG GPLGDNNQGA DGVGNASGDW HCDSTWMGDR VVTKSTRTWV LP SYNNHQY REIKSGSVDG SNANAYFGYS TPWGYFDFNR FHSHWSPRDW QRLINNYWGF RPRSLRVKIF NIQVKEVTVQ DST TTIANN LTSTVQVFTD DDYQLPYVVG NGTEGCLPAF PPQVFTLPQY GYATLNRDNT ENPTERSSFF CLEYFPSKML RTGN NFEFT YNFEEVPFHS SFAPSQNLFK LANPLVDQYL YRFVSTNNTG GVQFNKNLAG RYANTYKNWF PGPMGRTQGW NLGSG VNRA SVSAFATTNR MELEGASYQV PPQPNGMTNN LQGSNTYALE NTMIFNSQPA NPGTTATYLE GNMLITSESE TQPVNR VAY NVGGQMATNN QSSTTAPATG TYNLQEIVPG SVWMERDVYL QGPIWAKIPE TGAHFHPSPA MGGFGLKHPP PMMLIKN TP VPGNITSFSD VPVSSFITQY STGQVTVEME WELKKENSKR WNPEIQYTNN YNDPQFVDFA PDSTGEYRTT RPIGTRYL T RPL

UniProtKB: Capsid protein

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Macromolecule #2: DNA (5'-D(P*AP*A)-3')

MacromoleculeName: DNA (5'-D(P*AP*A)-3') / type: dna / ID: 2 / Number of copies: 60 / Classification: DNA
Source (natural)Organism: adeno-associated virus 5
Molecular weightTheoretical: 581.456 Da
SequenceString:
(DA)(DA)

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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

Concentration0.5 mg/mL
BufferpH: 7.4 / Component - Concentration: 1.0 mM / Component - Formula: PBS-MK / Component - Name: TD
Details: 10 mM Na2HPO4, 2 mM KH2PO4, 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, pH 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.967 µm / Nominal defocus min: 1.289 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3ntt

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 23000
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: COMMON LINE

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