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- PDB-28lw: CryoEM structure of carbon monoxide dehydrogenase from Ruminococc... -

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Basic information

Entry
Database: PDB / ID: 28lw
TitleCryoEM structure of carbon monoxide dehydrogenase from Ruminococcus flavefaciens
Componentsanaerobic carbon-monoxide dehydrogenase
KeywordsOXIDOREDUCTASE / carbon monoxide dehydrogenase / CODH / metalloenzyme / iron sulfur protein / anaerobic enzyme
Function / homologyIRON/SULFUR CLUSTER
Function and homology information
Biological speciesRuminococcus flavefaciens ATCC 19208 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.55 Å
AuthorsSrinivas, V. / Hogbom, M.
Funding support Sweden, Denmark, 3items
OrganizationGrant numberCountry
Knut and Alice Wallenberg Foundation2023.0201 Sweden
Swedish Research Council2017-04018 Sweden
Novo Nordisk FoundationNNF21OC0066716 Denmark
CitationJournal: Angew Chem Int Ed Engl / Year: 2026
Title: Beyond Canonical CO Oxidation: Structural and Evolutionary Insights Into a Non-Canonical Carbon Monoxide Dehydrogenase.
Authors: Maximilian Böhm / Vivek Srinivas / Benjamin Wiseman / Ping Huang / Moritz Senger / Martin Högbom / Henrik Land /
Abstract: Carbon monoxide dehydrogenases (CODHs) catalyse the reversible oxidation of CO to CO and play central roles in microbial carbon metabolism. While well-characterised CODHs from different phylogenetic ...Carbon monoxide dehydrogenases (CODHs) catalyse the reversible oxidation of CO to CO and play central roles in microbial carbon metabolism. While well-characterised CODHs from different phylogenetic backgrounds exhibit high bidirectional activity, the enigmatic clade B remains functionally uncharacterised. Here, we present the first structural and biochemical characterisation of a clade B CODH from Ruminococcus flavefaciens (RfCODH). It reveals striking divergence from canonical enzymes. A new anaerobic cryo-EM workflow was developed, carried out entirely under anoxic conditions by manual blotting and plunge freezing. It resulted in a 2.53 Å RfCODH structure. The structure adopts the typical CODH fold, but exhibits blocked gas channels, a compromised proton transfer pathway and disrupted cofactor coordination. This provides a structural rationale for RfCODH's severely attenuated CO oxidation activity (13 mU/mg vs. 900 U/mg for the well-studied ChCODH-II). EPR spectroscopy reveals unique oxidised C-cluster states not previously characterised in CODHs. Mirror tree analysis hints to co-evolution between clade B CODHs and associated ABC transporter substrate-binding proteins, suggesting these enzymes function in metabolism of substrates imported via the ABC transporter module. All findings indicate evolutionary repurposing of the CODH scaffold for alternative physiological functions.
History
DepositionFeb 6, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 23, 2026Provider: repository / Type: Initial release
Revision 1.0Sep 23, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: anaerobic carbon-monoxide dehydrogenase
B: anaerobic carbon-monoxide dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)159,0095
Polymers157,9552
Non-polymers1,0553
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 anaerobic carbon-monoxide dehydrogenase


Mass: 78977.250 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ruminococcus flavefaciens ATCC 19208 (bacteria)
Gene: SAMN04487860_10698 / Production host: Escherichia coli (E. coli) / References: anaerobic carbon monoxide dehydrogenase
#2: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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: Homodimer of carbon-monoxide dehydrogenase from Ruminococcus flavefaciens
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.158 MDa / Experimental value: NO
Source (natural)Organism: Ruminococcus flavefaciens ATCC 19208 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 50 mM Tris-HCl pH 8, 20 mM NaCl, 5 % w/v glycerol,
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 0 %
Details: Vetrification carried out by manually plunge-freezing in a nitrogen glove box

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Image recordingElectron dose: 41.65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCV4.7.1particle selection
4cryoSPARCV4.7.1CTF correction
7UCSF ChimeraX1.9model fitting
9cryoSPARCV4.7.1initial Euler assignment
10cryoSPARCV4.7.1final Euler assignment
12cryoSPARCV4.7.13D reconstruction
13PHENIX2.0_5885model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 123492 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 2.55 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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