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- EMDB-64143: Cryo-EM structure of ClassIII Salivaricin modification enzyme Sal... -

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Basic information

Entry
Database: EMDB / ID: EMD-64143
TitleCryo-EM structure of ClassIII Salivaricin modification enzyme SalKC in the presence of SalA
Map data
Sample
  • Complex: Dimeric structure of Class III lanthipeptide modification SalKC with AGS and SalA bound to one protomer.
    • Protein or peptide: SalKC
    • Protein or peptide: SalA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsLanthibiotic / RiPPs / LanKC / CryoEM / Antimicrobial peptides / ANTIMICROBIAL PROTEIN
Biological speciesStreptococcus salivarius (bacteria)
Methodsingle particle reconstruction / Resolution: 2.96 Å
AuthorsLi Y / Luo M / Shao K
Funding support Singapore, 1 items
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, 2025-
Header (metadata) releaseSep 3, 2025-
Map releaseSep 3, 2025-
UpdateOct 1, 2025-
Current statusOct 1, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_64143.png

Downloads & links

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Map

FileDownload / File: emd_64143.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.9548688 - 2.732432
Average (Standard dev.)-0.00051519653 (±0.061918903)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_64143_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_64143_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimeric structure of Class III lanthipeptide modification SalKC w...

EntireName: Dimeric structure of Class III lanthipeptide modification SalKC with AGS and SalA bound to one protomer.
Components
  • Complex: Dimeric structure of Class III lanthipeptide modification SalKC with AGS and SalA bound to one protomer.
    • Protein or peptide: SalKC
    • Protein or peptide: SalA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Dimeric structure of Class III lanthipeptide modification SalKC w...

SupramoleculeName: Dimeric structure of Class III lanthipeptide modification SalKC with AGS and SalA bound to one protomer.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Streptococcus salivarius (bacteria)
Molecular weightTheoretical: 210 KDa

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Macromolecule #1: SalKC

MacromoleculeName: SalKC / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus salivarius (bacteria)
Molecular weightTheoretical: 100.188383 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQYDFSLKDP LFFTLTDDDL TDFSSKYSAP LPYDWHELSN NSEWVNQYPI GFTTERQGWK VHISSDYKHS HEVLEVVSKV CHEFRVVFK YLKTEKVFVL RNGKNIDRGY SGKFITCYPN IESLEGFLKE LERKLKGYTG PYILSDRRWR EAPIYLRYGV F RESIPELE ...String:
MQYDFSLKDP LFFTLTDDDL TDFSSKYSAP LPYDWHELSN NSEWVNQYPI GFTTERQGWK VHISSDYKHS HEVLEVVSKV CHEFRVVFK YLKTEKVFVL RNGKNIDRGY SGKFITCYPN IESLEGFLKE LERKLKGYTG PYILSDRRWR EAPIYLRYGV F RESIPELE GNLKSDELLV SGKIIKDIRS PKFIIPEGLE IPEFLEKWLK DIKGDEKSEF PFTIESAIRF SNCGGIYNAT LS SSNKKII LREARPYTGL DFSGEYSTER MKSERRALTI LKDIDGIPNV FWYGKLWEHN FLGVEKMDGI PLNHWLTKNY PLY DSKGKE KYLYRAKNIL KQLISIVERA HKHSVYHQDI HFGNILIDRS DRLSLIDWEQ VRFDNSKMVE QKMAAPGYGS WIDD YPSKI DWYGVKQIAH YLYFPLIEQT SLVLGYDQQT FKVAHRNFVE MGYSDTDIKN MEVIINALDN KCSTFDNLSE KKILK PCLN NLVINSKADI NDFASRLGKG LLTISDEWKR KYKNKRIFPV HYYGLKINQG IAFSDLGILL SYKKLIDLLS DKVDNS YEE IKNLAIQTAI AKFRDDSPGL LDGMSGTIWI IHELGEKQLA IDLFRKYYSE MLRKSSEKNI YSGTAGILLV GLYLISQ HN DLSIKELIIS DMNLFADNYL FNPSDFCKVG VGDTNSNDPY EADSGLLFGH TGIGWLFGEA YRYTKNSKFL KCLNLAIE S ELVAYEKDDF DRLQYNQGKR LLPYLSTGSA GLLLLISRNK EFLQNSVLNN CHYLEKAVSP NFCVFPGIAN GMCGLFLSK NLYKSNINYV QQCKELIRCL ETYLCVVEDG FALAGDSGLK LTTDISTGTA GIILTLVSLR NGKLELLPSV Q

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Macromolecule #2: SalA

MacromoleculeName: SalA / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus salivarius (bacteria)
Molecular weightTheoretical: 1.792164 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MLEEVLKLQL MDAKE

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Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 2 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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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

Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationFormulaName
200.0 mMNaClsodium chloride
25.0 mM(HOCH2)3CNH2Tris

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 32.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing #1

Image processing ID1
Particle selectionNumber selected: 2440874
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 493047
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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Image processing #2

Image processing ID2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 493047
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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