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- PDB-8xmh: Potassium transporter KtrAB from Bacillus subtilis in ATP-bound s... -

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Basic information

Entry
Database: PDB / ID: 8xmh
TitlePotassium transporter KtrAB from Bacillus subtilis in ATP-bound state with addition of EDTA and EGTA, vertical C2 symmetry axis
Components
  • Ktr system potassium uptake protein A
  • Ktr system potassium uptake protein B
KeywordsTRANSPORT PROTEIN / KtrAB / RCK / potassium / transporter
Function / homology
Function and homology information


potassium:chloride symporter activity / monoatomic cation transmembrane transporter activity / potassium ion transport / identical protein binding / plasma membrane
Similarity search - Function
TrkH potassium transport family / Cation transporter / Cation transport protein / : / Regulator of K+ conductance, N-terminal / TrkA-N domain / Regulator of K+ conductance, C-terminal / Regulator of K+ conductance, C-terminal domain superfamily / TrkA-C domain / RCK C-terminal domain profile. ...TrkH potassium transport family / Cation transporter / Cation transport protein / : / Regulator of K+ conductance, N-terminal / TrkA-N domain / Regulator of K+ conductance, C-terminal / Regulator of K+ conductance, C-terminal domain superfamily / TrkA-C domain / RCK C-terminal domain profile. / RCK N-terminal domain profile. / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / : / Ktr system potassium uptake protein A / Ktr system potassium uptake protein B
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsChang, Y.K. / Chiang, W.T. / Hu, N.J. / Tsai, M.D.
Funding support Taiwan, 1items
OrganizationGrant numberCountry
Academia Sinica (Taiwan) Taiwan
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis and synergism of ATP and Na activation in bacterial K uptake system KtrAB.
Authors: Wesley Tien Chiang / Yao-Kai Chang / Wei-Han Hui / Shu-Wei Chang / Chen-Yi Liao / Yi-Chuan Chang / Chun-Jung Chen / Wei-Chen Wang / Chien-Chen Lai / Chun-Hsiung Wang / Siou-Ying Luo / Ya- ...Authors: Wesley Tien Chiang / Yao-Kai Chang / Wei-Han Hui / Shu-Wei Chang / Chen-Yi Liao / Yi-Chuan Chang / Chun-Jung Chen / Wei-Chen Wang / Chien-Chen Lai / Chun-Hsiung Wang / Siou-Ying Luo / Ya-Ping Huang / Shan-Ho Chou / Tzyy-Leng Horng / Ming-Hon Hou / Stephen P Muench / Ren-Shiang Chen / Ming-Daw Tsai / Nien-Jen Hu /
Abstract: The K uptake system KtrAB is essential for bacterial survival in low K environments. The activity of KtrAB is regulated by nucleotides and Na. Previous studies proposed a putative gating mechanism of ...The K uptake system KtrAB is essential for bacterial survival in low K environments. The activity of KtrAB is regulated by nucleotides and Na. Previous studies proposed a putative gating mechanism of KtrB regulated by KtrA upon binding to ATP or ADP. However, how Na activates KtrAB and the Na binding site remain unknown. Here we present the cryo-EM structures of ATP- and ADP-bound KtrAB from Bacillus subtilis (BsKtrAB) both solved at 2.8 Å. A cryo-EM density at the intra-dimer interface of ATP-KtrA was identified as Na, as supported by X-ray crystallography and ICP-MS. Thermostability assays and functional studies demonstrated that Na binding stabilizes the ATP-bound BsKtrAB complex and enhances its K flux activity. Comparing ATP- and ADP-BsKtrAB structures suggests that BsKtrB Arg417 and Phe91 serve as a channel gate. The synergism of ATP and Na in activating BsKtrAB is likely applicable to Na-activated K channels in central nervous system.
History
DepositionDec 27, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 3, 2024Provider: repository / Type: Initial release
Revision 1.1May 15, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.title / _citation.year
Revision 1.2May 22, 2024Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ktr system potassium uptake protein A
B: Ktr system potassium uptake protein A
C: Ktr system potassium uptake protein A
D: Ktr system potassium uptake protein A
E: Ktr system potassium uptake protein A
F: Ktr system potassium uptake protein A
G: Ktr system potassium uptake protein A
H: Ktr system potassium uptake protein A
I: Ktr system potassium uptake protein B
J: Ktr system potassium uptake protein B
K: Ktr system potassium uptake protein B
L: Ktr system potassium uptake protein B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)397,52628
Polymers393,22012
Non-polymers4,30616
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Ktr system potassium uptake protein A / K(+)-uptake protein KtrA


Mass: 24916.760 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: ktrA, yuaA, BSU31090 / Production host: Escherichia coli (E. coli) / References: UniProt: O32080
#2: Protein
Ktr system potassium uptake protein B / K(+)-uptake protein KtrB


Mass: 48471.539 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: ktrB, yubG, BSU31100 / Production host: Escherichia coli (E. coli) / References: UniProt: O32081
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Bacillus subtilis KtrAB potassium transporter KtrABCOMPLEXKtrAB complex(chain A-L). KtrAB complex in solution was composed of one KtrA octamer (chain A-H) and two KtrB dimer (chain I-J and chain K-L). The molecule weight of KtrAB complex (chain A-L) is 0.393 MDa#1-#20RECOMBINANT
2KtrA octamerCOMPLEXKtrA ocatmer (chain A-H) The molecule weight of KtrA octamer (chain A-H) is 0.199 MDa.#11RECOMBINANT
3KtrB dimerCOMPLEXTwo KtrB dimers (chain I-J and chain K-L) The molecule weight of each KtrB dimer is 0.097 MDa#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.393 MDaNO
210.199 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Bacillus subtilis (bacteria)1423
32Bacillus subtilis (bacteria)1423
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
Buffer solutionpH: 8
Details: 20 mM Tris-HCl pH 8.0, 70 mM NaCl, 30 mM KCl, 0.75 mM 6-cyclohexyl-1-hexyl-beta-D-maltoside, 2 mM EDTA, 1 mM EGTA, 1 mM ATP
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1400 nm
Image recordingAverage exposure time: 2 sec. / Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8613

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Processing

EM software
IDNameVersionCategory
7UCSF Chimera1.14model fitting
9PHENIX1.19.2model refinement
13cryoSPARC3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 527427 / Symmetry type: POINT
Atomic model buildingPDB-ID: 4J7C

4j7c


Accession code: 4J7C / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00327452
ELECTRON MICROSCOPYf_angle_d0.50737260
ELECTRON MICROSCOPYf_dihedral_angle_d9.3599808
ELECTRON MICROSCOPYf_chiral_restr0.0434484
ELECTRON MICROSCOPYf_plane_restr0.0044576

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