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- EMDB-60562: Cryo-EM structure of uropathogenic Escherichia coli CysK:CdiA:tRN... -

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Basic information

Entry
Database: EMDB / ID: EMD-60562
TitleCryo-EM structure of uropathogenic Escherichia coli CysK:CdiA:tRNA complex B
Map dataPostprocessed by deepEMhancer.
Sample
  • Complex: CysK in complex with CdiA-CT and tRNA.
    • Protein or peptide: Cysteine synthase A
    • Protein or peptide: tRNA nuclease CdiA
    • RNA: tRNAIleGAU
  • Ligand: MAGNESIUM ION
KeywordsRNase / Complex / Contact-dependent growth inhibition / Toxin
Function / homology
Function and homology information


tRNA-specific ribonuclease activity / cysteine synthase / cysteine synthase activity / other organism cell membrane / cysteine biosynthetic process from serine / RNA endonuclease activity / toxin activity / Hydrolases; Acting on ester bonds / host cell cytoplasm / tRNA binding ...tRNA-specific ribonuclease activity / cysteine synthase / cysteine synthase activity / other organism cell membrane / cysteine biosynthetic process from serine / RNA endonuclease activity / toxin activity / Hydrolases; Acting on ester bonds / host cell cytoplasm / tRNA binding / lyase activity / extracellular region / membrane
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. / : / Pyridoxal-phosphate dependent enzyme / Pyridoxal-phosphate dependent enzyme / Tryptophan synthase beta subunit-like PLP-dependent enzyme / Pectin lyase fold / Pectin lyase fold/virulence factor
Similarity search - Domain/homology
Cysteine synthase A / tRNA nuclease CdiA
Similarity search - Component
Biological speciesEscherichia coli 536 (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsFeng Z / Yashiro Y / Tomita K
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)18H03980 Japan
Japan Society for the Promotion of Science (JSPS)23H00368 Japan
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Mechanism of activation of contact-dependent growth inhibition tRNase toxin by the amino acid biogenesis factor CysK in the bacterial competition system.
Authors: Zhaohang Feng / Yuka Yashiro / Kozo Tomita /
Abstract: Contact-dependent growth inhibition (CDI) is a bacterial competition mechanism, wherein the C-terminal toxin domain of CdiA protein (CdiA-CT) is transferred from one bacterium to another, impeding ...Contact-dependent growth inhibition (CDI) is a bacterial competition mechanism, wherein the C-terminal toxin domain of CdiA protein (CdiA-CT) is transferred from one bacterium to another, impeding the growth of the toxin recipient. In uropathogenic Escherichia coli 536, CdiA-CT (CdiA-CTEC536) is a tRNA anticodon endonuclease that requires a cysteine biogenesis factor, CysK, for its activity. However, the mechanism underlying tRNA recognition and cleavage by CdiA-CTEC536 remains unresolved. Here, we present the cryo-EM structure of the CysK:CdiA-CTEC536:tRNA ternary complex. The interaction between CdiA-CTEC536 and CysK stabilizes the CdiA-CTEC536 structure and facilitates tRNA binding and the formation of the CdiA-CTEC536 catalytic core structure. The bottom-half of the tRNA interacts exclusively with CdiA-CTEC536 and the α-helices of CdiA-CTEC536 engage with the minor and major grooves of the bottom-half of tRNA, positioning the tRNA anticodon loop at the CdiA-CTEC536 catalytic site for tRNA cleavage. Thus, CysK serves as a platform facilitating the recognition and cleavage of substrate tRNAs by CdiA-CTEC536.
History
DepositionJun 17, 2024-
Header (metadata) releaseAug 21, 2024-
Map releaseAug 21, 2024-
UpdateMar 5, 2025-
Current statusMar 5, 2025Processing site: PDBj / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_60562.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed by deepEMhancer.
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.0018045262 - 1.6805198
Average (Standard dev.)0.00068520126 (±0.019203324)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 278.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : CysK in complex with CdiA-CT and tRNA.

EntireName: CysK in complex with CdiA-CT and tRNA.
Components
  • Complex: CysK in complex with CdiA-CT and tRNA.
    • Protein or peptide: Cysteine synthase A
    • Protein or peptide: tRNA nuclease CdiA
    • RNA: tRNAIleGAU
  • Ligand: MAGNESIUM ION

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Supramolecule #1: CysK in complex with CdiA-CT and tRNA.

SupramoleculeName: CysK in complex with CdiA-CT and tRNA. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia coli 536 (bacteria)
Molecular weightTheoretical: 110 KDa

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Macromolecule #1: Cysteine synthase A

MacromoleculeName: Cysteine synthase A / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: cysteine synthase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 34.756688 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSKIFEDNSL TIGHTPLVRL NRIGNGRILA KVESRNPSFS V(LLP)CRIGANMI WDAEKRGVLK PGVELVEPTS GNTGIA LAY VAAARGYKLT LTMPETMSIE RRKLLKALGA NLVLTEGAKG MKGAIQKAEE IVASNPEKYL LLQQFSNPAN PEIHEKT TG PEIWEDTDGQ ...String:
MSKIFEDNSL TIGHTPLVRL NRIGNGRILA KVESRNPSFS V(LLP)CRIGANMI WDAEKRGVLK PGVELVEPTS GNTGIA LAY VAAARGYKLT LTMPETMSIE RRKLLKALGA NLVLTEGAKG MKGAIQKAEE IVASNPEKYL LLQQFSNPAN PEIHEKT TG PEIWEDTDGQ VDVFIAGVGT GGTLTGVSRY IKGTKGKTDL ISVAVEPTDS PVIAQALAGE EIKPGPHKIQ GIGAGFIP A NLDLKLVDKV IGITNEEAIS TARRLMEEEG ILAGISSGAA VAAALKLQED ESFTNKNIVV ILPSSGERYL STALFADLF TEKELQQ

UniProtKB: Cysteine synthase A

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Macromolecule #2: tRNA nuclease CdiA

MacromoleculeName: tRNA nuclease CdiA / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Escherichia coli 536 (bacteria)
Molecular weightTheoretical: 25.18125 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHVEN NALSLVARGC AVAAPCRTKV AEQLLEIGAK AGMAGLAGAA VKDMADRMTS DELEHLITLQ MMGNDEITTK YLSSLHDKY GSGAASNPNI GKDLTDAEKV ELGGSGSGTG TPPPSENDPK QQNEKTVDKL NQKQESAIKK IDNTIKNALK D HDIIGTLK ...String:
MHHHHHHVEN NALSLVARGC AVAAPCRTKV AEQLLEIGAK AGMAGLAGAA VKDMADRMTS DELEHLITLQ MMGNDEITTK YLSSLHDKY GSGAASNPNI GKDLTDAEKV ELGGSGSGTG TPPPSENDPK QQNEKTVDKL NQKQESAIKK IDNTIKNALK D HDIIGTLK DMDGKPVPKE NGGYWDAMQE MQNTLRGLRN HADTLKNVNN PEAQAAYGRA TDAINKIESA LKGYGI

UniProtKB: tRNA nuclease CdiA

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Macromolecule #3: tRNAIleGAU

MacromoleculeName: tRNAIleGAU / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 25.000869 KDa
SequenceString:
AGGCUUGUAG CUCAGGUGGU UAGAGCGCAC CCCUGAU(T6A)AG GGUGAGGUCG GUGGUUCAAG UCCACUCAGG CCUACC A

GENBANK: GENBANK: CP053605.1

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.6 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
25.0 mMTris-HClTris-HCl
50.0 mMNaClSodium chloride
2.0 mMMgCl2Magnesium chloride
10.0 mM2-mercaptoethanol2-mercaptoethanol

Details: 25mM Tris-HCl,50mM NaCl,2mM MgCl2, 10mM 2-mercaptoethanol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 4092 pixel / Digitization - Dimensions - Height: 5760 pixel / Number grids imaged: 1 / Number real images: 7044 / Average exposure time: 1.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4707496
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 103127
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

5j43


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-8zyd:
Cryo-EM structure of uropathogenic Escherichia coli CysK:CdiA:tRNA complex B

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