+Open data
-Basic information
Entry | Database: PDB / ID: 8hf6 | ||||||
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Title | Cryo-EM structure of nucleotide-bound ComA E647Q mutant | ||||||
Components | Competence factor transporting ATP-binding protein/permease ComA | ||||||
Keywords | HYDROLASE / TRANSLOCASE / ABC transporter / PCAT / MEMBRANE PROTEIN / TRANSPORT PROTEIN | ||||||
Function / homology | Function and homology information ABC-type bacteriocin transporter activity / ATPase-coupled lipid transmembrane transporter activity / cysteine-type peptidase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / ATP hydrolysis activity / proteolysis / ATP binding / membrane Similarity search - Function | ||||||
Biological species | Streptococcus pneumoniae D39 (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | ||||||
Authors | Yu, L. / Xin, X. / Min, L. | ||||||
Funding support | Singapore, 1items
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Citation | Journal: Nat Commun / Year: 2023 Title: Structural basis of peptide secretion for Quorum sensing by ComA. Authors: Lin Yu / Xin Xu / Wan-Zhen Chua / Hao Feng / Zheng Ser / Kai Shao / Jian Shi / Yumei Wang / Zongli Li / Radoslaw M Sobota / Lok-To Sham / Min Luo / Abstract: Quorum sensing (QS) is a crucial regulatory mechanism controlling bacterial signalling and holds promise for novel therapies against antimicrobial resistance. In Gram-positive bacteria, such as ...Quorum sensing (QS) is a crucial regulatory mechanism controlling bacterial signalling and holds promise for novel therapies against antimicrobial resistance. In Gram-positive bacteria, such as Streptococcus pneumoniae, ComA is a conserved efflux pump responsible for the maturation and secretion of peptide signals, including the competence-stimulating peptide (CSP), yet its structure and function remain unclear. Here, we functionally characterize ComA as an ABC transporter with high ATP affinity and determined its cryo-EM structures in the presence or absence of CSP or nucleotides. Our findings reveal a network of strong electrostatic interactions unique to ComA at the intracellular gate, a putative binding pocket for two CSP molecules, and negatively charged residues facilitating CSP translocation. Mutations of these residues affect ComA's peptidase activity in-vitro and prevent CSP export in-vivo. We demonstrate that ATP-Mg triggers the outward-facing conformation of ComA for CSP release, rather than ATP alone. Our study provides molecular insights into the QS signal peptide secretion, highlighting potential targets for QS-targeting drugs. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8hf6.cif.gz | 207.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8hf6.ent.gz | 163.4 KB | Display | PDB format |
PDBx/mmJSON format | 8hf6.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8hf6_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
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Full document | 8hf6_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 8hf6_validation.xml.gz | 40.5 KB | Display | |
Data in CIF | 8hf6_validation.cif.gz | 60 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/hf/8hf6 ftp://data.pdbj.org/pub/pdb/validation_reports/hf/8hf6 | HTTPS FTP |
-Related structure data
Related structure data | 34714MC 8hf4C 8hf5C 8hf7C 8k4bC 8k7aC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 80432.039 Da / Num. of mol.: 2 / Mutation: E647Q Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus pneumoniae D39 (bacteria) Gene: comA, AZJ70_04775, ERS019166_01078, GM529_06210, GM538_05890, GM546_02340 Production host: Escherichia coli (E. coli) References: UniProt: A0A0B7KN15, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases #2: Chemical | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: ComA / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Source (natural) | Organism: Streptococcus pneumoniae D39 (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 / Details: 25 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT |
Specimen | Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293.15 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 1.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
EM software | Name: PHENIX / Version: 1.19.2_4158: / Category: model refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84972 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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