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- EMDB-15894: KimA from B. subtilis with nucleotide second-messenger c-di-AMP bound -

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Basic information

Entry
Database: EMDB / ID: EMD-15894
TitleKimA from B. subtilis with nucleotide second-messenger c-di-AMP bound
Map data
Sample
  • Complex: KimA with bound c-di-AMP
    • Protein or peptide: Potassium transporter KimA
  • Ligand: (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
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: POTASSIUM ION
Keywordspotassium importer / second-messenger / c-di-AMP / MEMBRANE PROTEIN
Function / homology: / Amino acid/polyamine transporter I / Amino acid permease / symporter activity / potassium ion transport / metal ion binding / plasma membrane / Potassium transporter KimA
Function and homology information
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsVonck J / Wieferig JP
Funding support Germany, 2 items
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 MF / Wieferig JP / Corey R / Hellmich Y / Tascon I / Sousa JS / Stansfeld P / Vonck J / Haenelt I
History
DepositionSep 28, 2022-
Header (metadata) releaseJan 18, 2023-
Map releaseJan 18, 2023-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15894.png

Downloads & links

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Map

FileDownload / File: emd_15894.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 264 pix.
= 220.968 Å		0.84 Å/pix.
x 264 pix.
= 220.968 Å		0.84 Å/pix.
x 264 pix.
= 220.968 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.837 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.024
Minimum - Maximum-0.106560916 - 0.16826828
Average (Standard dev.)0.0003060819 (±0.0059163724)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 220.968 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpened map

Fileemd_15894_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_15894_half_map_1.map
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Histogram

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Half map: #1

Fileemd_15894_half_map_2.map
Projections & Slices
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Sample components

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Entire : KimA with bound c-di-AMP

EntireName: KimA with bound c-di-AMP
Components
  • Complex: KimA with bound c-di-AMP
    • Protein or peptide: Potassium transporter KimA
  • Ligand: (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
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: POTASSIUM ION

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Supramolecule #1: KimA with bound c-di-AMP

SupramoleculeName: KimA with bound c-di-AMP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 134 KDa

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Macromolecule #1: Potassium transporter KimA

MacromoleculeName: Potassium transporter KimA / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 66.838977 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MYHSIKRFLI GKPLKSQAAG EQKLTKLKAL AMLSSDALSS VAYGTEQILI ILATISAAAF WYSIPIAVGV LILLLALILS YRQIIYAYP QGGGAYIVSK ENLGEKPGLI AGGSLLVDYI LTVAVSISAG TDAITSAFPA LHDYHVPIAI FLVLVIMILN L RGLSESAS ...String:
MYHSIKRFLI GKPLKSQAAG EQKLTKLKAL AMLSSDALSS VAYGTEQILI ILATISAAAF WYSIPIAVGV LILLLALILS YRQIIYAYP QGGGAYIVSK ENLGEKPGLI AGGSLLVDYI LTVAVSISAG TDAITSAFPA LHDYHVPIAI FLVLVIMILN L RGLSESAS ILAYPVYLFV VALLVLIAVG LFKLMTGQID QPAHHTSLGT PVAGITLFLL LKAFSSGCSA LTGVEAISNA IP AFKNPPA RNAARTLAMM GILLAILFSG ITVLAYGYGT APKPDETVVS QIASETFGRN VFYYVIQGVT SLILVLAANT GFS AFPQLA FNLARDQYMP RMFTVRGDRL GFSNGIIFLG FASIVLIILF GGQTEHLIPL YAVGVFIPFT LSQTGMCMKW IKQK PKGWI GKMLINSCGA LISFMVLSIL FVTKFNVVWP VLIFMPIVVL LFFAIKNHYT AVGEQLRIVD KEPEEIKGTV VIVPV AGVT TVVQKSIHYA KSLSDQVIAV HVSFDREQEK KFEKRWEELN NGVRLVTLHS SYRSLVHPFD KFLETVEAKA KKEQFS VMV LFPQFITKKR WHTILHNQSA FLLRVRLFWK KDIMVATLPY HFKK

UniProtKB: Potassium transporter KimA

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Macromolecule #2: (2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-2,9-bis(6-amino-9H-purin-...

MacromoleculeName: (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
type: ligand / ID: 2 / Number of copies: 2 / Formula: 2BA
Molecular weightTheoretical: 658.412 Da

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Macromolecule #3: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 3 / Number of copies: 7 / Formula: LMT
Molecular weightTheoretical: 510.615 Da
Chemical component information

ChemComp-LMT:
DODECYL-BETA-D-MALTOSIDE / detergent*YM

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Macromolecule #4: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 4 / Number of copies: 2 / 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

Concentration4 mg/mL
BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.1 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

10092

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 332000
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6s3k


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

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

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