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- PDB-8b70: KimA from B. subtilis with nucleotide second-messenger c-di-AMP bound -

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Basic information

Entry
Database: PDB / ID: 8b70
TitleKimA from B. subtilis with nucleotide second-messenger c-di-AMP bound
ComponentsPotassium transporter KimA
KeywordsMEMBRANE PROTEIN / potassium importer / second-messenger / c-di-AMP
Function / homologyAmino 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 speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsVonck, J. / Wieferig, J.P.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)HA 6322/4-1 Germany
German Research Foundation (DFG)VO 1449/1-1 Germany
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
DepositionSep 28, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 18, 2023Provider: repository / Type: Initial release
Revision 2.0May 17, 2023Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Polymer sequence / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / em_3d_fitting_list ...atom_site / em_3d_fitting_list / entity / entity_name_com / entity_poly / entity_src_gen / pdbx_contact_author / pdbx_entity_instance_feature / pdbx_entry_details / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_close_contact / pdbx_validate_torsion / struct_asym / struct_conf / struct_conn / struct_ref / struct_ref_seq / struct_sheet_range
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.source_name ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity_name_com.name / _entity_poly.pdbx_strand_id / _entity_src_gen.pdbx_gene_src_gene / _pdbx_entry_details.has_ligand_of_interest / _pdbx_poly_seq_scheme.pdb_strand_id / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.value / _pdbx_struct_sheet_hbond.range_1_auth_asym_id / _pdbx_struct_sheet_hbond.range_1_auth_comp_id / _pdbx_struct_sheet_hbond.range_1_auth_seq_id / _pdbx_struct_sheet_hbond.range_1_label_comp_id / _pdbx_struct_sheet_hbond.range_1_label_seq_id / _pdbx_struct_sheet_hbond.range_2_auth_asym_id / _pdbx_struct_sheet_hbond.range_2_auth_comp_id / _pdbx_struct_sheet_hbond.range_2_auth_seq_id / _pdbx_struct_sheet_hbond.range_2_label_comp_id / _pdbx_struct_sheet_hbond.range_2_label_seq_id / _pdbx_unobs_or_zero_occ_residues.auth_asym_id / _pdbx_validate_torsion.auth_asym_id / _pdbx_validate_torsion.phi / _pdbx_validate_torsion.psi / _struct_conf.beg_auth_asym_id / _struct_conf.end_auth_asym_id / _struct_conf.end_auth_comp_id / _struct_conf.end_auth_seq_id / _struct_conf.end_label_comp_id / _struct_conf.end_label_seq_id / _struct_conf.pdbx_PDB_helix_length / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref_seq.pdbx_db_accession / _struct_ref_seq.pdbx_strand_id / _struct_sheet_range.beg_auth_asym_id / _struct_sheet_range.end_auth_asym_id
Description: Atoms with unrealistic or zero occupancies / Details: Removed side chains without density / Provider: author / Type: Coordinate replacement
Revision 2.1Jul 5, 2023Group: Database references / Category: citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Potassium transporter KimA
B: Potassium transporter KimA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)138,64713
Polymers133,6782
Non-polymers4,96911
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14830 Å2
ΔGint-44 kcal/mol
Surface area45730 Å2
MethodPISA

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Components

#1: Protein Potassium transporter KimA / K(+) importer A / Potassium-proton symporter KimA


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): LB 2003 / References: UniProt: P96589
#2: Chemical ChemComp-2BA / (2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-2,9-bis(6-amino-9H-purin-9-yl)octahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8 ]tetraoxadiphosphacyclododecine-3,5,10,12-tetrol 5,12-dioxide / bis-(3',5')-cyclic-dimeric-Adenosine-monophosphate


Mass: 658.412 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C20H24N10O12P2
#3: Sugar
ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM
#4: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K
Has ligand of interestN

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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: KimA with bound c-di-AMP / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.134 MDa / Experimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: LB2003
Buffer solutionpH: 8
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1100 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1Topazparticle selection
2EPUimage acquisition
4GctfCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 332000 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6S3K

6s3k


Accession code: 6S3K / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0069482
ELECTRON MICROSCOPYf_angle_d0.7212890
ELECTRON MICROSCOPYf_dihedral_angle_d20.6681308
ELECTRON MICROSCOPYf_chiral_restr0.0441586
ELECTRON MICROSCOPYf_plane_restr0.0051504

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