+Open data
-Basic information
Entry | Database: PDB / ID: 8b71 | |||||||||
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Title | Upright KimA dimer with bound c-di-AMP from B. subtilis | |||||||||
Components | Potassium transporter KimA | |||||||||
Keywords | MEMBRANE PROTEIN / potassium importer / second messenger / c-di-AMP | |||||||||
Function / homology | Amino acid/polyamine transporter I / Amino acid permease / symporter activity / potassium ion transport / metal ion binding / plasma membrane / Chem-2BA / Potassium transporter KimA Function and homology information | |||||||||
Biological species | Bacillus subtilis (bacteria) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å | |||||||||
Authors | Vonck, J. / Wieferig, J.P. | |||||||||
Funding support | Germany, 2items
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Citation | Journal: Nat Commun / Year: 2023 Title: Cyclic di-AMP traps proton-coupled K transporters of the KUP family in an inward-occluded conformation. Authors: Michael F Fuss / Jan-Philip Wieferig / Robin A Corey / Yvonne Hellmich / Igor Tascón / Joana S Sousa / Phillip J Stansfeld / Janet Vonck / Inga Hänelt / Abstract: Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls ...Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K/H symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K concentrations, further K accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K binding site and therefore traps KimA in an inward-occluded conformation. #1: Journal: Biorxiv / Year: 2023 Title: Cyclic di-AMP traps proton-coupled K+ transporters of the KUP family in an inward-occluded conformation Authors: Fuss, M.F. / Wieferig, J.P. / Corey, R. / Hellmich, Y. / Tascon, I. / Sousa, J.S. / Stansfeld, P. / Vonck, J. / Haenelt, I. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8b71.cif.gz | 194.4 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8b71.ent.gz | 157.5 KB | Display | PDB format |
PDBx/mmJSON format | 8b71.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/b7/8b71 ftp://data.pdbj.org/pub/pdb/validation_reports/b7/8b71 | HTTPS FTP |
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-Related structure data
Related structure data | 15895MC 8b70C C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 66838.977 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: kimA, ydaO, BSU04320 / Production host: Escherichia coli (E. coli) / Strain (production host): LB2003 / References: UniProt: P96589 #2: Chemical | Has ligand of interest | N | |
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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: Upright dimer KimA with c-di-AMP bound / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Value: 0.134 MDa / Experimental value: NO |
Source (natural) | Organism: Bacillus subtilis (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: LB2003 |
Buffer solution | pH: 8 |
Specimen | Conc.: 2.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Reconstituted in amphipols PMAL C8 |
Specimen support | Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 |
Vitrification | Cryogen name: ETHANE |
-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: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1100 nm |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 55 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 296000 / Symmetry type: POINT | ||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6S3K Accession code: 6S3K / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||
Refine LS restraints |
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