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- PDB-9ugq: Cryo-EM structure of ClassIII Salivaricin modification enzyme Sal... -

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Basic information

Entry
Database: PDB / ID: 9ugq
TitleCryo-EM structure of ClassIII Salivaricin modification enzyme SalKC in the presence of SalA
Components
  • SalA
  • SalKC
KeywordsANTIMICROBIAL PROTEIN / Lanthibiotic / RiPPs / LanKC / CryoEM / Antimicrobial peptides
Function / homologyPHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
Function and homology information
Biological speciesStreptococcus salivarius (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / Resolution: 2.96 Å
AuthorsLi, Y. / Luo, M. / Shao, K. / Li, Y.
Funding support Singapore, 1items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore) Singapore
CitationJournal: ACS Chem Biol / Year: 2025
Title: Structural Basis of Substrate Recognition and Nucleotide Specificity in the Class III-b LanKC Enzyme SalKC.
Authors: Yifan Li / Kai Shao / Yicong Li / Bee Koon Gan / Min Luo /
Abstract: Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) with potent antimicrobial functions. Their biosynthesis is carried out by dedicated biosynthetic enzymes, ...Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) with potent antimicrobial functions. Their biosynthesis is carried out by dedicated biosynthetic enzymes, including the recently described Class III-b LanKC enzymes, which represent a newly defined subclass of trifunctional synthetases. Here, we report the high-resolution cryo-EM structure and biochemical characterization of SalKC from , which catalyzes the maturation of the antimicrobial peptide salivaricin. SalKC adopts a conserved dimeric architecture stabilized by a His36 hotspot, mirroring that of the previously characterized PneKC. Cryo-EM structure resolved to sub-3.0 Å revealed the side chains of the bound leader peptide in atomic detail, allowing clear visualization of a conserved recognition motif and offering new structural insight into peptide engagement. Biochemical assays showed that SalKC prefers ATP over GTP, contrasting with the GTP-preferring PneKC. Structural comparison identified a single amino acid switch: Lys303 in SalKC versus His300 in PneKC, as the key determinant of this specificity. Mutation of Lys303 to histidine reverses nucleotide preference, confirming its functional role. Together, these findings revealed conserved principles and specialized adaptations within Class III-b LanKC enzymes and provided a molecular framework for understanding their substrate and cofactor selectivity.
History
DepositionApr 13, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 3, 2025Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Sep 10, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.2Oct 1, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: SalKC
B: SalKC
E: SalA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)203,2647
Polymers202,1693
Non-polymers1,0954
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 SalKC


Mass: 100188.383 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus salivarius (bacteria) / Production host: Escherichia coli (E. coli)
#2: Protein/peptide SalA


Mass: 1792.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus salivarius (bacteria) / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Dimeric structure of Class III lanthipeptide modification SalKC with AGS and SalA bound to one protomer.
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.21 MDa / Experimental value: YES
Source (natural)Organism: Streptococcus salivarius (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
225 mMTris(HOCH2)3CNH21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 32 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
Image processing
IDImage recording-ID
11
21
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
IDImage processing-IDNum. of particles selected
112440874
22
3D reconstruction
IDResolution (Å)Resolution methodNum. of particlesImage processing-IDEntry-IDSymmetry type
12.96FSC 0.143 CUT-OFF49304719UGQPOINT
22.96FSC 0.143 CUT-OFF49304719UGQPOINT
32.96FSC 0.143 CUT-OFF49304719UGQPOINT
42.96FSC 0.143 CUT-OFF49304719UGQPOINT
52.96FSC 0.143 CUT-OFF49304729UGQPOINT
62.96FSC 0.143 CUT-OFF49304729UGQPOINT
72.96FSC 0.143 CUT-OFF49304729UGQPOINT
82.96FSC 0.143 CUT-OFF49304729UGQPOINT
RefinementHighest resolution: 2.96 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413968
ELECTRON MICROSCOPYf_angle_d0.86618945
ELECTRON MICROSCOPYf_dihedral_angle_d13.7484982
ELECTRON MICROSCOPYf_chiral_restr0.0512100
ELECTRON MICROSCOPYf_plane_restr0.0062380

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