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- EMDB-60564: Cryo-EM structure of uropathogenic Escherichia coli 2:2 CysK:CdiA... -

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Basic information

Entry
Database: EMDB / ID: EMD-60564
TitleCryo-EM structure of uropathogenic Escherichia coli 2:2 CysK:CdiA complex
Map data
Sample
  • Complex: 2 CysK in complex with 2 CdiA-CT.
    • Protein or peptide: CysK
    • Protein or peptide: CdiA-CT
KeywordsRNase / Complex / Contact-dependent growth inhibition / Toxin
Biological speciesEscherichia coli 536 (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.78 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

Supplemental images

emd_60564.png

Downloads & links

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Map

FileDownload / File: emd_60564.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 212.48 Å		0.83 Å/pix.
x 256 pix.
= 212.48 Å		0.83 Å/pix.
x 256 pix.
= 212.48 Å

Surface

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Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.21844515 - 0.4586711
Average (Standard dev.)-0.00012009538 (±0.013532069)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_60564_msk_1.map
Projections & Slices
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Half map: #1

Fileemd_60564_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_60564_half_map_2.map
Projections & Slices
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Sample components

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Entire : 2 CysK in complex with 2 CdiA-CT.

EntireName: 2 CysK in complex with 2 CdiA-CT.
Components
  • Complex: 2 CysK in complex with 2 CdiA-CT.
    • Protein or peptide: CysK
    • Protein or peptide: CdiA-CT

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Supramolecule #1: 2 CysK in complex with 2 CdiA-CT.

SupramoleculeName: 2 CysK in complex with 2 CdiA-CT. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli 536 (bacteria)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: CysK

MacromoleculeName: CysK / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
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

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Macromolecule #2: CdiA-CT

MacromoleculeName: CdiA-CT / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli 536 (bacteria)
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

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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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 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: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.78 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 290703
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5j43


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

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