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- PDB-7vyv: Cryo-EM structure of Depo32, a Klebsiella phage depolymerase targ... -

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Basic information

Entry
Database: PDB / ID: 7vyv
TitleCryo-EM structure of Depo32, a Klebsiella phage depolymerase targets the K2 serotype K. pneumoniae
ComponentsDepolymerase
KeywordsLYASE / Depolymerase / Klebsiella pneumoniae / K2 capsular type / Capsular polysaccharides
Function / homologybiological process involved in interaction with host / Pectin lyase fold/virulence factor / viral life cycle / virion component / Depolymerase
Function and homology information
Biological speciesKlebsiella phage GH-K3 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.32 Å
AuthorsCai, R. / Ren, Z. / Zhao, R. / Wang, X. / Guo, Z. / Du, R. / Han, W. / Ru, H. / Gu, J.
Funding support China, 3items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32072824 China
National Natural Science Foundation of China (NSFC)31872505 China
National Natural Science Foundation of China (NSFC)U19A2038 China
CitationJournal: To Be Published
Title: Depo32, a depolymerase of Klebsiella phage, efficiently controls infection caused by Klebsiella pneumoniae with K2 serotype CPS
Authors: Cai, R. / Ren, Z. / Zhao, R. / Wang, X. / Guo, Z. / Du, R. / Han, W. / Ru, H. / Gu, J.
History
DepositionNov 15, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 30, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Depolymerase
B: Depolymerase
C: Depolymerase


Theoretical massNumber of molelcules
Total (without water)295,9363
Polymers295,9363
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Depolymerase / Depo32


Mass: 98645.289 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella phage GH-K3 (virus) / Gene: GHK3_32 / Plasmid: pET28a(+) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A3S7W7I3

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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: Tail spike protein Depo32 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)Organism: Klebsiella phage 020009 (virus) / Strain: vB_KpnS_GH-K3
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3) / Plasmid: pET28a(+)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris(hydroxymethyl)aminomethaneNH2C(CH2OH)31
2150 mMsodium chlorideNaCl1
30.5 mMtris(2-carboxyethyl)phosphineC9H15O6P1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 285.15 K / Details: blot 3.5 to 4 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DARK FIELD / Calibrated magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.56 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: (1.18.2_3874: ???) / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
4RELIONCTF correction
7UCSF Chimeramodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 462388 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 2.32→278.27 Å / SU ML: 0.32 / σ(F): 0.63 / Phase error: 37.53 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rwork0.3308 --
obs0.3308 3613319 99.98 %
Rfree-2016 6 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00917112
ELECTRON MICROSCOPYf_angle_d1.08723238
ELECTRON MICROSCOPYf_dihedral_angle_d15.7322337
ELECTRON MICROSCOPYf_chiral_restr0.0662520
ELECTRON MICROSCOPYf_plane_restr0.0083051
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.32-2.380.5521440.5255258396ELECTRON MICROSCOPY100
2.38-2.440.57621560.5121257647ELECTRON MICROSCOPY100
2.44-2.510.52281080.5045257841ELECTRON MICROSCOPY100
2.51-2.60.51371320.5258091ELECTRON MICROSCOPY100
2.6-2.690.47381440.4854257480ELECTRON MICROSCOPY100
2.69-2.80.4561680.466258417ELECTRON MICROSCOPY100
2.8-2.920.42581320.4331258063ELECTRON MICROSCOPY100
2.92-3.080.3961440.3895258417ELECTRON MICROSCOPY100
3.08-3.270.3171320.328257661ELECTRON MICROSCOPY100
3.27-3.520.24561320.259258488ELECTRON MICROSCOPY100
3.52-3.880.20791680.2022257371ELECTRON MICROSCOPY100
3.88-4.440.16941440.1547257867ELECTRON MICROSCOPY100
4.44-5.590.13221320.1363258149ELECTRON MICROSCOPY100
5.59-278.270.17931800.1819257415ELECTRON MICROSCOPY100

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