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- PDB-9dcb: The Structure of AAV5 at 4 Degrees -

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Basic information

Entry
Database: PDB / ID: 9dcb
TitleThe Structure of AAV5 at 4 Degrees
Components
  • Capsid protein
  • DNA (5'-D(P*AP*A)-3')
KeywordsVIRUS / temperature / genome / vector / icosahedron
Function / homology
Function and homology information


T=1 icosahedral viral capsid / structural molecule activity
Similarity search - Function
Phospholipase 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
Similarity search - Domain/homology
DNA / Capsid protein
Similarity search - Component
Biological speciesadeno-associated virus 5
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsBennett, A.B. / McKenna, R.
Funding support United States, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 16, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Capsid protein
B: Capsid protein
C: Capsid protein
D: Capsid protein
E: Capsid protein
F: Capsid protein
G: Capsid protein
H: Capsid protein
I: Capsid protein
J: Capsid protein
K: Capsid protein
L: Capsid protein
M: Capsid protein
N: Capsid protein
O: Capsid protein
P: Capsid protein
Q: Capsid protein
R: Capsid protein
S: Capsid protein
T: Capsid protein
U: Capsid protein
V: Capsid protein
W: Capsid protein
X: Capsid protein
Y: Capsid protein
Z: Capsid protein
a: Capsid protein
b: Capsid protein
c: Capsid protein
d: Capsid protein
e: Capsid protein
f: Capsid protein
g: Capsid protein
h: Capsid protein
i: Capsid protein
j: Capsid protein
k: Capsid protein
l: Capsid protein
m: Capsid protein
n: Capsid protein
o: Capsid protein
p: Capsid protein
q: Capsid protein
r: Capsid protein
s: Capsid protein
t: Capsid protein
u: Capsid protein
v: Capsid protein
w: Capsid protein
x: Capsid protein
y: Capsid protein
z: Capsid protein
1: Capsid protein
2: Capsid protein
3: Capsid protein
4: Capsid protein
5: Capsid protein
6: Capsid protein
7: Capsid protein
8: Capsid protein
0: DNA (5'-D(P*AP*A)-3')
JA: DNA (5'-D(P*AP*A)-3')
UA: DNA (5'-D(P*AP*A)-3')
fA: DNA (5'-D(P*AP*A)-3')
qA: DNA (5'-D(P*AP*A)-3')
1A: DNA (5'-D(P*AP*A)-3')
3A: DNA (5'-D(P*AP*A)-3')
4A: DNA (5'-D(P*AP*A)-3')
5A: DNA (5'-D(P*AP*A)-3')
9: DNA (5'-D(P*AP*A)-3')
AA: DNA (5'-D(P*AP*A)-3')
BA: DNA (5'-D(P*AP*A)-3')
CA: DNA (5'-D(P*AP*A)-3')
DA: DNA (5'-D(P*AP*A)-3')
EA: DNA (5'-D(P*AP*A)-3')
FA: DNA (5'-D(P*AP*A)-3')
GA: DNA (5'-D(P*AP*A)-3')
HA: DNA (5'-D(P*AP*A)-3')
IA: DNA (5'-D(P*AP*A)-3')
KA: DNA (5'-D(P*AP*A)-3')
LA: DNA (5'-D(P*AP*A)-3')
MA: DNA (5'-D(P*AP*A)-3')
NA: DNA (5'-D(P*AP*A)-3')
OA: DNA (5'-D(P*AP*A)-3')
PA: DNA (5'-D(P*AP*A)-3')
QA: DNA (5'-D(P*AP*A)-3')
RA: DNA (5'-D(P*AP*A)-3')
SA: DNA (5'-D(P*AP*A)-3')
TA: DNA (5'-D(P*AP*A)-3')
VA: DNA (5'-D(P*AP*A)-3')
WA: DNA (5'-D(P*AP*A)-3')
XA: DNA (5'-D(P*AP*A)-3')
YA: DNA (5'-D(P*AP*A)-3')
ZA: DNA (5'-D(P*AP*A)-3')
aA: DNA (5'-D(P*AP*A)-3')
bA: DNA (5'-D(P*AP*A)-3')
cA: DNA (5'-D(P*AP*A)-3')
dA: DNA (5'-D(P*AP*A)-3')
eA: DNA (5'-D(P*AP*A)-3')
gA: DNA (5'-D(P*AP*A)-3')
hA: DNA (5'-D(P*AP*A)-3')
iA: DNA (5'-D(P*AP*A)-3')
jA: DNA (5'-D(P*AP*A)-3')
kA: DNA (5'-D(P*AP*A)-3')
lA: DNA (5'-D(P*AP*A)-3')
mA: DNA (5'-D(P*AP*A)-3')
nA: DNA (5'-D(P*AP*A)-3')
oA: DNA (5'-D(P*AP*A)-3')
pA: DNA (5'-D(P*AP*A)-3')
rA: DNA (5'-D(P*AP*A)-3')
sA: DNA (5'-D(P*AP*A)-3')
tA: DNA (5'-D(P*AP*A)-3')
uA: DNA (5'-D(P*AP*A)-3')
vA: DNA (5'-D(P*AP*A)-3')
wA: DNA (5'-D(P*AP*A)-3')
xA: DNA (5'-D(P*AP*A)-3')
yA: DNA (5'-D(P*AP*A)-3')
zA: DNA (5'-D(P*AP*A)-3')
0A: DNA (5'-D(P*AP*A)-3')
2A: DNA (5'-D(P*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)4,864,732120
Polymers4,864,732120
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Capsid protein


Mass: 80497.414 Da / Num. of mol.: 60
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) adeno-associated virus 5 / Gene: cap, VP1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9YIJ1
#2: DNA chain ...
DNA (5'-D(P*AP*A)-3')


Mass: 581.456 Da / Num. of mol.: 60
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) adeno-associated virus 5 / Production host: Spodoptera frugiperda (fall armyworm)
Has ligand of interestN
Has protein modificationN

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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 5 / Type: VIRUS / Details: Baculovirus expression / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 4 MDa / Experimental value: NO
Source (natural)Organism: adeno-associated virus 5
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Details of virusEmpty: NO / Enveloped: NO / Isolate: SEROTYPE / Type: VIRION
Buffer solutionpH: 7.4
Details: 10 mM Na2HPO4, 2 mM KH2PO4, 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, pH 7.4
Buffer componentConc.: 1 mM / Name: TD / Formula: PBS-MK
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3967 nm / Nominal defocus min: 1289 nm
Image recordingElectron dose: 45 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.10-2155_2155: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01257460
ELECTRON MICROSCOPYf_angle_d0.959352200
ELECTRON MICROSCOPYf_dihedral_angle_d9.619202080
ELECTRON MICROSCOPYf_chiral_restr0.06136660
ELECTRON MICROSCOPYf_plane_restr0.00846440

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