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- PDB-8zyc: Cryo-EM structure of uropathogenic Escherichia coli CysK:CdiA:tRN... -

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Basic information

Entry
Database: PDB / ID: 8zyc
TitleCryo-EM structure of uropathogenic Escherichia coli CysK:CdiA:tRNA complex A
Components
  • Cysteine synthase A
  • tRNA nuclease CdiA
  • tRNAIleGAU
KeywordsTOXIN / RNase / Complex / Contact-dependent growth inhibition
Function / homology
Function and homology information


tRNA-specific ribonuclease activity / cysteine synthase / L-cysteine desulfhydrase activity / cysteine synthase activity / other organism cell membrane / cysteine biosynthetic process from serine / RNA endonuclease activity / toxin activity / host cell cytoplasm / tRNA binding ...tRNA-specific ribonuclease activity / cysteine synthase / L-cysteine desulfhydrase activity / cysteine synthase activity / other organism cell membrane / cysteine biosynthetic process from serine / RNA endonuclease activity / toxin activity / host cell cytoplasm / tRNA binding / Hydrolases; Acting on ester bonds / extracellular region / membrane / cytoplasm
Similarity search - Function
Toxin CdiA-like, Filamentous hemagglutinin motif repeats / VENN motif-containing domain / Pre-toxin domain with VENN motif / Hemagglutinin repeat / Hemagglutinin repeat / Filamentous haemagglutinin FhaB/tRNA nuclease CdiA-like, TPS domain / TPS secretion domain / haemagglutination activity domain / Cysteine synthase CysK / Cysteine synthase ...Toxin CdiA-like, Filamentous hemagglutinin motif repeats / VENN motif-containing domain / Pre-toxin domain with VENN motif / Hemagglutinin repeat / Hemagglutinin repeat / Filamentous haemagglutinin FhaB/tRNA nuclease CdiA-like, TPS domain / TPS secretion domain / haemagglutination activity domain / Cysteine synthase CysK / Cysteine synthase / Parallel beta-helix repeat / Parallel beta-helix repeats / Cysteine synthase/cystathionine beta-synthase, pyridoxal-phosphate attachment site / Cysteine synthase/cystathionine beta-synthase P-phosphate attachment site. / : / Pectin lyase fold / Pyridoxal-phosphate dependent enzyme / Tryptophan synthase beta subunit-like PLP-dependent enzyme / Pyridoxal-phosphate dependent enzyme / Pectin lyase fold/virulence factor
Similarity search - Domain/homology
: / RNA / RNA (> 10) / Cysteine synthase A / tRNA nuclease CdiA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Escherichia coli 536 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsFeng, Z. / Yashiro, Y. / Tomita, K.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)18H03980 Japan
Japan Society for the Promotion of Science (JSPS)23H00368 Japan
CitationJournal: To Be Published
Title: Mechanism of activation of contact-dependent growth inhibition tRNase toxin by the amino acid biogenesis factor CysK in the bacterial competition system.
Authors: Feng, Z. / Yashiro, Y. / Tomita, K.
History
DepositionJun 17, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 21, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cysteine synthase A
B: tRNA nuclease CdiA
C: tRNAIleGAU
E: Cysteine synthase A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)119,7446
Polymers119,6954
Non-polymers492
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Cysteine synthase A / CSase A / O-acetylserine (thiol)-lyase A / OAS-TL A / O-acetylserine sulfhydrylase A / Sulfate ...CSase A / O-acetylserine (thiol)-lyase A / OAS-TL A / O-acetylserine sulfhydrylase A / Sulfate starvation-induced protein 5 / SSI5


Mass: 34756.688 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: cysK, Z3680, ECs3286 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0ABK6, cysteine synthase
#2: Protein tRNA nuclease CdiA / tRNase CdiA / Anticodon nuclease CdiA / CdiA-EC536 / Toxin CdiA


Mass: 25181.250 Da / Num. of mol.: 1 / Mutation: H178A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli 536 (bacteria) / Gene: cdiA, ECP_4580 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q0T963, Hydrolases; Acting on ester bonds
#3: RNA chain tRNAIleGAU


Mass: 25000.869 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / Strain (production host): JM101tr / References: GenBank: 1845258627
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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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: CysK in complex with CdiA-CT and tRNA. / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.11 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli 536 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7
Details: 25mM Tris-HCl,50mM NaCl,2mM MgCl2, 10mM 2-mercaptoethanol
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HClTris-HCl1
250 mMSodium chlorideNaCl1
32 mMMagnesium chlorideMgCl21
410 mM2-mercaptoethanol2-mercaptoethanol1
SpecimenConc.: 1.6 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 R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7044
Image scansWidth: 4092 / Height: 5760

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Processing

EM softwareName: PHENIX / Version: 1.18.2_3874: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4707496
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115994 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5J43

5j43


Accession code: 5J43 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0087317
ELECTRON MICROSCOPYf_angle_d0.83510260
ELECTRON MICROSCOPYf_dihedral_angle_d11.6662960
ELECTRON MICROSCOPYf_chiral_restr0.0541242
ELECTRON MICROSCOPYf_plane_restr0.011048

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