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- EMDB-36800: Potassium transporter KtrAB from Bacillus subtilis in ADP-bound state -

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Basic information

Entry
Database: EMDB / ID: EMD-36800
TitlePotassium transporter KtrAB from Bacillus subtilis in ADP-bound state
Map datamain map for KtrAB
Sample
  • Complex: Bacillus subtilis KtrAB potassium transporter
    • Complex: KtrA octamer
      • Protein or peptide: Ktr system potassium uptake protein A
    • Complex: KtrB dimer
      • Protein or peptide: Ktr system potassium uptake protein B
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: POTASSIUM ION
KeywordsKtrAB / RCK / potassium / transporter / TRANSPORT PROTEIN
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
Ktr system potassium uptake protein A / Ktr system potassium uptake protein B
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.83 Å
AuthorsChang YK / Chiang WT / Hu NJ / Tsai MD
Funding support Taiwan, 1 items
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
DepositionJul 11, 2023-
Header (metadata) releaseApr 3, 2024-
Map releaseApr 3, 2024-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36800.png

Downloads & links

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Map

FileDownload / File: emd_36800.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map for KtrAB
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 318.72 Å		0.83 Å/pix.
x 384 pix.
= 318.72 Å		0.83 Å/pix.
x 384 pix.
= 318.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-1.1169102 - 1.6550835
Average (Standard dev.)0.00020117348 (±0.036854316)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 318.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_36800_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: focused refinement map for KtrA

Fileemd_36800_additional_1.map
Annotationfocused refinement map for KtrA
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: focused refinement map for KtrB

Fileemd_36800_additional_2.map
Annotationfocused refinement map for KtrB
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: half map A for KtrAB

Fileemd_36800_half_map_1.map
Annotationhalf map A for KtrAB
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B for KtrAB

Fileemd_36800_half_map_2.map
Annotationhalf map B for KtrAB
Projections & Slices
AxesZYX

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Histogram

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Sample components

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Entire : Bacillus subtilis KtrAB potassium transporter

EntireName: Bacillus subtilis KtrAB potassium transporter
Components
  • Complex: Bacillus subtilis KtrAB potassium transporter
    • Complex: KtrA octamer
      • Protein or peptide: Ktr system potassium uptake protein A
    • Complex: KtrB dimer
      • Protein or peptide: Ktr system potassium uptake protein B
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: POTASSIUM ION

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Supramolecule #1: Bacillus subtilis KtrAB potassium transporter

SupramoleculeName: Bacillus subtilis KtrAB potassium transporter / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: KtrAB 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
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 199 KDa

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Supramolecule #2: KtrA octamer

SupramoleculeName: KtrA octamer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: KtrA ocatmer (chain A-H) The molecule weight of KtrA octamer (chain A-H) is 0.199 MDa.
Source (natural)Organism: Bacillus subtilis (bacteria)

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Supramolecule #3: KtrB dimer

SupramoleculeName: KtrB dimer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Details: Two KtrB dimers (chain I-J and chain K-L) The molecule weight of each KtrB dimer is 0.097 MDa

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Macromolecule #1: Ktr system potassium uptake protein A

MacromoleculeName: Ktr system potassium uptake protein A / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 24.91676 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGRIKNKQFA VIGLGRFGGS ICKELHRMGH EVLAVDINEE KVNAYASYAT HAVIANATEE NELLSLGIRN FEYVIVAIGA NIQASTLTT LLLKELDIPN IWVKAQNYYH HKVLEKIGAD RIIHPEKDMG VKIAQSLSDE NVLNYIDLSD EYSIVELLAT R KLDSKSII ...String:
MGRIKNKQFA VIGLGRFGGS ICKELHRMGH EVLAVDINEE KVNAYASYAT HAVIANATEE NELLSLGIRN FEYVIVAIGA NIQASTLTT LLLKELDIPN IWVKAQNYYH HKVLEKIGAD RIIHPEKDMG VKIAQSLSDE NVLNYIDLSD EYSIVELLAT R KLDSKSII DLNVRAKYGC TILAIKHHGD ICLSPAPEDI IREQDCLVIM GHKKDIKRFE NEGM

UniProtKB: Ktr system potassium uptake protein A

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Macromolecule #2: Ktr system potassium uptake protein B

MacromoleculeName: Ktr system potassium uptake protein B / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 48.471539 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTLQKDKVIK WVRFTPPQVL AIGFFLTIII GAVLLMLPIS TTKPLSWIDA LFTAASATTV TGLAVVDTGT QFTVFGQTVI MGLIQIGGL GFMTFAVLIV MILGKKIGLK ERMLVQEALN QPTIGGVIGL VKVLFLFSIS IELIAALILS IRLVPQYGWS S GLFASLFH ...String:
MTLQKDKVIK WVRFTPPQVL AIGFFLTIII GAVLLMLPIS TTKPLSWIDA LFTAASATTV TGLAVVDTGT QFTVFGQTVI MGLIQIGGL GFMTFAVLIV MILGKKIGLK ERMLVQEALN QPTIGGVIGL VKVLFLFSIS IELIAALILS IRLVPQYGWS S GLFASLFH AISAFNNAGF SLWPDNLMSY VGDPTVNLVI TFLFITGGIG FTVLFDVMKN RRFKTFSLHT KLMLTGTLML NA IAMLTVF ILEYSNPGTL GHLHIVDKLW ASYFQAVTPR TAGFNSLDFG SMREGTIVFT LLLMFIGAGS ASTASGIKLT TFI VILTSV IAYLRGKKET VIFRRSIKYP IIIKALAVSV TSLFIVFLGI FALTITEQAP FLQIVFETFS AFGTVGLTMG LTPE LTTAG KCIIIVIMFI GRIGPLTFVF SFAKTEQSNI RYPDGEVFTG

UniProtKB: Ktr system potassium uptake protein B

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 8 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #4: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.24 mg/mL
BufferpH: 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, 0.1 mM ADP
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 6919 / Average exposure time: 2.5 sec. / Average electron dose: 36.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 294844
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

4j7c

source_name: PDB, initial_model_type: experimental modelKtrB region

5but

source_name: PDB, initial_model_type: experimental modelKtrA region
Output model

PDB-8k1s:
Potassium transporter KtrAB from Bacillus subtilis in ADP-bound state

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