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- PDB-6e9d: Sub-2 Angstrom Ewald Curvature-Corrected Single-Particle Cryo-EM ... -

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Basic information

Entry
Database: PDB / ID: 6e9d
TitleSub-2 Angstrom Ewald Curvature-Corrected Single-Particle Cryo-EM Reconstruction of AAV-2 L336C
ComponentsCapsid protein VP1
KeywordsVIRUS LIKE PARTICLE / AAV-2 L336C / Gene therapy / Ewald sphere curvature correction / Per particle CTF
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 Srivastava/1982
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.86 Å
AuthorsTan, Y.Z. / Aiyer, S. / Mietzsch, M. / Hull, J.A. / McKenna, R. / Baker, T.S. / Agbandje-McKenna, M. / Lyumkis, D.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI136680-01 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM109524 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM033050 United States
CitationJournal: Nat Commun / Year: 2018
Title: Sub-2 Å Ewald curvature corrected structure of an AAV2 capsid variant.
Authors: Yong Zi Tan / Sriram Aiyer / Mario Mietzsch / Joshua A Hull / Robert McKenna / Joshua Grieger / R Jude Samulski / Timothy S Baker / Mavis Agbandje-McKenna / Dmitry Lyumkis /
Abstract: Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures ...Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we exploit several technical advances to improve resolution, including per-particle contrast transfer function (CTF) refinement and correction for Ewald sphere curvature. The latter is demonstrated with several experimental samples and should become more standard as resolutions increase or at lower microscope accelerating voltages. The combined application of the described methods to micrographs recorded on a Titan Krios enables structure determination at ~1.86-Å resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle. The resulting structural details provide an improved model for understanding the biology of AAV that will guide future vector development for gene therapy.
History
DepositionJul 31, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 15, 2018Provider: repository / Type: Initial release
Revision 1.1Sep 19, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Feb 26, 2020Group: Source and taxonomy / Category: entity_src_gen
Item: _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Structure viewerMolecule:
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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
1: Capsid protein VP1
2: Capsid protein VP1
3: Capsid protein VP1
4: Capsid protein VP1
5: Capsid protein VP1
6: 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
7: Capsid protein VP1
8: Capsid protein VP1


Theoretical massNumber of molelcules
Total (without water)4,921,28060
Polymers4,921,28060
Non-polymers00
Water212,93711820
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area935590 Å2
ΔGint-3762 kcal/mol
Surface area792190 Å2

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Components

#1: Protein ...
Capsid protein VP1 /


Mass: 82021.336 Da / Num. of mol.: 60 / Mutation: L336C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus 2 Srivastava/1982
Gene: VP1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P03135
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 11820 / Source method: isolated from a natural source / Formula: H2O

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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: #1 / Source: RECOMBINANT
Molecular weightValue: 3.9 MDa / Experimental value: NO
Source (natural)Organism: Adeno-associated virus - 2
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9 / Plasmid: pFastBac1-HM
Details of virusEmpty: YES / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Homo sapiens
Virus shellName: Icosahedral capsidCapsid / Diameter: 250 nm / Triangulation number (T number): 1
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
22 mMmagnesium chlorideMgCl21
350 mMHEPESC8H18N2O4S1
SpecimenConc.: 1.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 277 K
Details: Double blotting was used to increase particle concentration. Sample was added to a plasma-cleaned (Gatan Solarus) grid and blotted away using Whatman grade 4 filter paper after 20 second wait time.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Beam tilt in 8 directions +/- 5 mrad
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 37000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.5 sec. / Electron dose: 22.5 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1317 / Details: Stage shift mode for data collection
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 70 / Used frames/image: 5-19

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Processing

EM software
IDNameVersionCategory
1Gautomatch0.56particle selection
2Leginonimage acquisition
4RELION2.1CTF correction
7Coot0.8.9.1model fitting
9PHENIX1.13model refinement
10cryoSPARC0.65initial Euler assignment
11FREALIGN9.11final Euler assignment
12FREALIGN9.11classification
13FREALIGN9.113D reconstruction
Image processingDetails: super-resolution mode
CTF correctionDetails: CTF estimation per particle - Gctf, CTF estimation per micrograph - CTFFIND4
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 78194
Details: Template-based picking by generating template from a 2-D classification of an initial subset of data.
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 1.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30515 / Algorithm: FOURIER SPACE / Details: Frequency limited refinement / Symmetry type: POINT
Atomic model buildingB value: 61.9 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient, EM ringer score
Atomic model buildingPDB-ID: 1LP3

1lp3


Accession code: 1LP3 / Source name: PDB / Type: experimental model

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