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- EMDB-70168: CryoEM structure of EcKatG S-Trp105 at 2.22 Angstrom resolution r... -

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Basic information

Entry
Database: EMDB / ID: EMD-70168
TitleCryoEM structure of EcKatG S-Trp105 at 2.22 Angstrom resolution revealing an asymmetric sulfur center in O=S-Trp
Map dataDensity modified map at 2.22 angstrom resolution
Sample
  • Complex: EcKatG S-Trp105
    • Protein or peptide: Catalase-peroxidase
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: water
KeywordsMet-Tyr-Trp cofactor / heme-dependent enzyme / non-canonical amino acid / OXIDOREDUCTASE
Function / homology
Function and homology information


catalase-peroxidase / catalase activity / hydrogen peroxide catabolic process / cellular response to hydrogen peroxide / heme binding / metal ion binding / cytosol
Similarity search - Function
Catalase-peroxidase haem / Peroxidases heam-ligand binding site / Peroxidase, active site / Peroxidases active site signature. / Plant heme peroxidase family profile. / Haem peroxidase / Peroxidase / Peroxidases proximal heme-ligand signature. / Haem peroxidase superfamily
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.22 Å
AuthorsDuan R / Li J / Nathan B / Yang X / Liu A
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM152982 United States
CitationJournal: Nat Commun / Year: 2026
Title: Single-atom substitution redirects KatG reactivity from cofactor biogenesis to stereoselective sulfoxidation.
Authors: Ran Duan / Jiasong Li / Wendell P Griffith / Yang Xu / Nathan D Burrows / Anthony P Green / Aimin Liu /
Abstract: Protein-derived cofactors rely on precisely positioned heteroatoms to direct redox chemistry, yet isolating their individual contributions remains challenging. The indole N-H of tryptophan plays a ...Protein-derived cofactors rely on precisely positioned heteroatoms to direct redox chemistry, yet isolating their individual contributions remains challenging. The indole N-H of tryptophan plays a central yet elusive role in biogenesis and function of the Met-Tyr-Trp (MYW) cofactor in catalase-peroxidase (KatG). Here, we use genetic code expansion to replace cofactor-forming Trp105 with thiotryptophan (S-Trp), enabling a single-heteroatom (N → S) substitution. Instead of forming the MYW crosslink, KatG bearing S-Trp105 undergoes site-specific monooxygenation to yield a chiral sulfoxide. HPLC-MS, circular dichroism, and FT-IR spectroscopy identify selective oxygen insertion at the sulfur, establishing enantioselective formation of an (S)-configured sulfoxide. A 2.22 Å cryo-EM structure visualizes the oxidized S-Trp105, revealing the S = O moiety orienting toward the iron and confirming the absence of crosslinking. The S-atom oxygenation is heme-dependent and proceeds via a two-electron oxygen-atom transfer, contrasting with the radical-mediated one-electron chemistry of native tryptophan. This redirection suppresses catalase activity by perturbing cofactor formation. These results show that a single-atom substitution reroutes the distal heme site from radical crosslinking to stereoselective sulfoxidation, uncovering a monooxygenase-like capability within KatG. This work highlights using noncanonical amino acids to achieve atomic-level control over reaction pathways and to interrogate cofactor biogenesis with unprecedented precision.
History
DepositionApr 11, 2025-
Header (metadata) releaseApr 22, 2026-
Map releaseApr 22, 2026-
UpdateAug 19, 2026-
Current statusAug 19, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70168.png

Downloads & links

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Map

FileDownload / File: emd_70168.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity modified map at 2.22 angstrom resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.95 Å/pix.
x 240 pix.
= 228.96 Å		0.95 Å/pix.
x 240 pix.
= 228.96 Å		0.95 Å/pix.
x 240 pix.
= 228.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.954 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.36
Minimum - Maximum-2.960021 - 4.785946
Average (Standard dev.)0.00006406984 (±0.15663265)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 228.95999 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map from cryoSPARC

Fileemd_70168_additional_1.map
AnnotationUnsharpened map from cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unsharpened half-map from cryoSPARC

Fileemd_70168_half_map_1.map
AnnotationUnsharpened half-map from cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unsharpened half-map from cryoSPARC

Fileemd_70168_half_map_2.map
AnnotationUnsharpened half-map from cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : EcKatG S-Trp105

EntireName: EcKatG S-Trp105
Components
  • Complex: EcKatG S-Trp105
    • Protein or peptide: Catalase-peroxidase
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: water

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Supramolecule #1: EcKatG S-Trp105

SupramoleculeName: EcKatG S-Trp105 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: KatG protein with 3-benzothienyl-L-alanine (S-Trp) genetically incorporated in place of W105
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 320 KDa

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Macromolecule #1: Catalase-peroxidase

MacromoleculeName: Catalase-peroxidase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: catalase-peroxidase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 81.105672 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSTSDDIHNT TATGKCPFHQ GGHDQSAGAG TTTRDWWPNQ LRVDLLNQHS NRSNPLGEDF DYRKEFSKLD YYGLKKDLKA LLTESQPWW PADWGSYAGL FIRMA(A1B9V)HGAG TYRSIDGRGG AGRGQQRFAP LNSWPDNVSL DKARRLLWPI KQKYG QKIS ...String:
MSTSDDIHNT TATGKCPFHQ GGHDQSAGAG TTTRDWWPNQ LRVDLLNQHS NRSNPLGEDF DYRKEFSKLD YYGLKKDLKA LLTESQPWW PADWGSYAGL FIRMA(A1B9V)HGAG TYRSIDGRGG AGRGQQRFAP LNSWPDNVSL DKARRLLWPI KQKYG QKIS WADLFILAGN VALENSGFRT FGFGAGREDV WEPDLDVNWG DEKAWLTHRH PEALAKAPLG ATEMGLIYVN PEGPDH SGE PLSAAAAIRA TFGNMGMNDE ETVALIAGGH TLGKTHGAGP TSNVGPDPEA APIEEQGLGW ASTYGSGVGA DAITSGL EV VWTQTPTQWS NYFFENLFKY EWVQTRSPAG AIQFEAVDAP EIIPDPFDPS KKRKPTMLVT DLTLRFDPEF EKISRRFL N DPQAFNEAFA RAWFKLTHRD MGPKSRYIGP EVPKEDLIWQ DPLPQPIYNP TEQDIIDLKF AIADSGLSVS ELVSVAWAS ASTFRGGDKR GGANGARLAL MPQRDWDVNA AAVRALPVLE KIQKESGKAS LADIIVLAGV VGVEKAASAA GLSIHVPFAP GRVDARQDQ TDIEMFELLE PIADGFRNYR ARLDVSTTES LLIDKAQQLT LTAPEMTALV GGMRVLGANF DGSKNGVFTD R VGVLSNDF FVNLLDMRYE WKATDESKEL FEGRDRETGE VKFTASRADL VFGSNSVLRA VAEVYASSDA HEKFVKDFVA AW VKVMNLD RFDLLEHHHH HH

UniProtKB: Catalase-peroxidase

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Macromolecule #2: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 2 / Number of copies: 4 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 20 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.12 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mM(HOCH2)3CNH2Tris(hydroxymethyl)aminomethane
50.0 mMNaClSodium chloride

Details: 50 mM Tris-HCl, 50 mM NaCl at pH 8
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.026000000000000002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3 microliter of the sample was applied to each grid and blotted for 3 s.
DetailsThe KatG S-Trp105 was dissolved in the 50 mM Tris-HCl, 50 mM NaCl buffer at pH 8

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average exposure time: 6.58 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 / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 7762366 / Details: Particles were selected from blob picking.
CTF correctionSoftware - Name: cryoSPARC (ver. 4.5.3) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 43 / Applied symmetry - Point group: D4 (2x4 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.3) / Number images used: 884126
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3)
Final 3D classificationNumber classes: 200 / Software - Name: cryoSPARC (ver. 4.5.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7jz6


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 7.92
Output model

PDB-9o6a:
CryoEM structure of EcKatG S-Trp105 at 2.22 Angstrom resolution revealing an asymmetric sulfur center in O=S-Trp

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