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- PDB-30bt: Cryo-EM structure of Beta-lactamase-like domain from Neomoorella ... -

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Basic information

Entry
Database: PDB / ID: 30bt
TitleCryo-EM structure of Beta-lactamase-like domain from Neomoorella carbonis
ComponentsBeta-lactamase-like domain
KeywordsMEMBRANE PROTEIN / DNA translocation / Nuclease / DNA binding
Biological speciesNeomoorella carbonis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsDeselaers, S. / Wang, D. / Cairoli, T. / Afanasyev, P. / Hospenthal, M.K.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Swiss State Secretariat for Education, Research and InnovationMB22.00043 Switzerland
Swiss National Science Foundation3200-0-239918 Switzerland
CitationJournal: Nucleic Acids Res / Year: 2026
Title: Structure and biochemistry reveal substrate-modulated ComEC nuclease activity during DNA processing.
Authors: Sophie Deselaers / Dianhong Wang / Tamino Cairoli / Pavel Afanasyev / Manuela K Hospenthal /
Abstract: Natural transformation enables bacteria to internalize extracellular DNA, driving adaptation and the spread of antibiotic resistance. The membrane protein ComEC mediates translocation of single- ...Natural transformation enables bacteria to internalize extracellular DNA, driving adaptation and the spread of antibiotic resistance. The membrane protein ComEC mediates translocation of single-stranded DNA (ssDNA) across the cytoplasmic membrane while degrading the complementary strand, yet the structural basis of its activity remains incompletely defined. Here, we report a cryo-electron microscopy structure of full-length ComEC from Neomoorella carbonis in a pre-translocation state, revealing a three-domain architecture and a conserved transmembrane channel captured in a closed conformation. Structural analysis indicates that conformational rearrangements of channel-lining helices would be required to accommodate ssDNA. Biochemical assays show that, relative to the isolated β-lactamase-like domain, full-length ComEC degrades DNA more efficiently and exhibits position-dependent cleavage of phosphodiester bonds within the DNA substrate. Importantly, coating of the DNA by the periplasmic DNA receptor ComEA suppresses endonucleolytic cleavage and enhances 5'' terminal cleavage, thereby directing ComEC towards productive processing of transforming DNA during natural transformation.
History
DepositionApr 20, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 20, 2026Provider: repository / Type: Initial release
Revision 1.0May 20, 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: Beta-lactamase-like domain


Theoretical massNumber of molelcules
Total (without water)87,6651
Polymers87,6651
Non-polymers00
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 Beta-lactamase-like domain


Mass: 87664.914 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neomoorella carbonis (bacteria) / Production host: Escherichia coli (E. coli)
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: Beta-lactamase-like domain / Type: COMPLEX / Details: ComEC solublized in LMNG / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.087 MDa / Experimental value: NO
Source (natural)Organism: Neomoorella carbonis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 0.01% (w/v) LMNG was added during affinity purification but excluded during SEC.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES-NaOH1
250 mMSodium ChlorideNaCl1
32 mMBeta-mercaptoethanolBeta-mercaptoethanol1
SpecimenConc.: 5 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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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: 165000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 1100 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.5particle selection
2EPU3.10.0image acquisition
4cryoSPARC4.7.1CTF correction
7UCSF ChimeraX1.11.1model fitting
9cryoSPARC4.7.1initial Euler assignment
10cryoSPARC4.7.1final Euler assignment
12cryoSPARC4.7.13D reconstruction
13PHENIX1.21.2-5419model refinement
14Rosetta3.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3276527
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 383072 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 4.2 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0022147
ELECTRON MICROSCOPYf_angle_d0.4142922
ELECTRON MICROSCOPYf_dihedral_angle_d8.928789
ELECTRON MICROSCOPYf_chiral_restr0.046320
ELECTRON MICROSCOPYf_plane_restr0.004391

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