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- PDB-5ipk: Structure of the R432A variant of Adeno-associated virus type 2 VLP -

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Basic information

Entry
Database: PDB / ID: 5ipk
TitleStructure of the R432A variant of Adeno-associated virus type 2 VLP
ComponentsCapsid protein VP1
KeywordsVIRUS LIKE PARTICLE / Adeno-associated virus / R432A / gene therapy / icosahedral / dependoparvovirus
Function / homology
Function and homology information


permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner membrane / host cell nucleolus / T=1 icosahedral viral capsid / clathrin-dependent endocytosis of virus by host cell / structural molecule activity / virion attachment to host cell
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
Biological speciesAdeno-associated virus - 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsDrouin, L.M. / Lins, B. / Janssen, M.E. / Bennet, A. / Chipman, P. / McKenna, R. / Chen, W. / Muzyczka, N. / Cardone, G. / Baker, T.S. / Agbandje-McKenna, M.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM33050 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL51811 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI1081961 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32-GM008799 United States
CitationJournal: J Virol / Year: 2016
Title: Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging.
Authors: Lauren M Drouin / Bridget Lins / Maria Janssen / Antonette Bennett / Paul Chipman / Robert McKenna / Weijun Chen / Nicholas Muzyczka / Giovanni Cardone / Timothy S Baker / Mavis Agbandje-McKenna /
Abstract: The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector ...The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency.
IMPORTANCE: The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism ...IMPORTANCE: The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ∼1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.
History
DepositionMar 9, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 20, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2016Group: Database references
Revision 1.2Sep 28, 2016Group: Database references
Revision 1.3Sep 13, 2017Group: Author supporting evidence / Database references / Category: citation / pdbx_audit_support
Item: _citation.journal_id_CSD / _pdbx_audit_support.funding_organization
Revision 1.4Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Capsid protein VP1
B: Capsid protein VP1
C: Capsid protein VP1
D: Capsid protein VP1
E: Capsid protein VP1
F: Capsid protein VP1
G: Capsid protein VP1
H: Capsid protein VP1
I: Capsid protein VP1
J: Capsid protein VP1
K: Capsid protein VP1
L: Capsid protein VP1
M: Capsid protein VP1
N: Capsid protein VP1
O: Capsid protein VP1
P: Capsid protein VP1
Q: Capsid protein VP1
R: Capsid protein VP1
S: Capsid protein VP1
T: Capsid protein VP1
U: Capsid protein VP1
V: Capsid protein VP1
W: Capsid protein VP1
X: Capsid protein VP1
Y: Capsid protein VP1
Z: Capsid protein VP1
a: Capsid protein VP1
b: Capsid protein VP1
c: Capsid protein VP1
d: Capsid protein VP1
e: Capsid protein VP1
f: Capsid protein VP1
g: Capsid protein VP1
h: Capsid protein VP1
i: Capsid protein VP1
j: Capsid protein VP1
k: Capsid protein VP1
l: Capsid protein VP1
m: Capsid protein VP1
n: Capsid protein VP1
o: Capsid protein VP1
p: Capsid protein VP1
q: Capsid protein VP1
r: Capsid protein VP1
s: Capsid protein VP1
t: Capsid protein VP1
u: Capsid protein VP1
v: Capsid protein VP1
w: Capsid protein VP1
x: Capsid protein VP1
y: Capsid protein VP1
z: Capsid protein VP1
1: Capsid protein VP1
2: Capsid protein VP1
3: Capsid protein VP1
4: Capsid protein VP1
5: Capsid protein VP1
6: Capsid protein VP1
7: Capsid protein VP1
8: Capsid protein VP1


Theoretical massNumber of molelcules
Total (without water)4,916,71460
Polymers4,916,71460
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area842640 Å2
ΔGint-3919 kcal/mol
Surface area998650 Å2

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Components

#1: Protein ...
Capsid protein VP1 /


Mass: 81945.234 Da / Num. of mol.: 60 / Mutation: R432A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus - 2 / Gene: VP1 / Production host: unidentified baculovirus / References: UniProt: P03135

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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 - 2 / Type: VIRUS / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Adeno-associated virus - 2
Source (recombinant)Organism: unidentified baculovirus / Plasmid: pFBDVPm11
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Homo sapiens
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESHEPES1
2100 mMsodium chlorideNaClSodium chloride1
31 mMcalcium chlorideCaCl21
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: Grid was blotted for 5 seconds before plunge freezing.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 56924 X / Nominal defocus max: 3200 nm / Nominal defocus min: 1200 nm / Cs: 2.26 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER / Temperature (max): 97 K / Temperature (min): 90 K
Image recordingAverage exposure time: 0.8 sec. / Electron dose: 20 e/Å2 / Film or detector model: KODAK SO-163 FILM / Num. of grids imaged: 1 / Num. of real images: 49
Image scansSampling size: 6.35 µm

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Processing

SoftwareName: PHENIX / Version: 1.10pre_2097: / Classification: refinement
EM software
IDNameVersionCategory
1RobEMv4.0particle selection
4CTFFINDV3CTF correction
12Auto3DEMv4.053D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 19457
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 19457 / Symmetry type: POINT
RefinementHighest resolution: 3.7 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.011247620
ELECTRON MICROSCOPYf_angle_d1.252337680
ELECTRON MICROSCOPYf_dihedral_angle_d9.057198120
ELECTRON MICROSCOPYf_chiral_restr0.06535400
ELECTRON MICROSCOPYf_plane_restr0.00844820

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