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- EMDB-15895: Upright KimA dimer with bound c-di-AMP from B. subtilis -

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Basic information

Entry
Database: EMDB / ID: EMD-15895
TitleUpright KimA dimer with bound c-di-AMP from B. subtilis
Map data
Sample
  • Complex: Upright dimer KimA with c-di-AMP bound
    • 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
Keywordspotassium importer / second messenger / c-di-AMP / MEMBRANE PROTEIN
Function / homologyAmino 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: 3.8 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 5, 2023-
Current statusJul 5, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15895.png

Downloads & links

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Map

FileDownload / File: emd_15895.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.831 Å
Density
Contour LevelBy AUTHOR: 0.017
Minimum - Maximum-0.12849109 - 0.17800094
Average (Standard dev.)0.00009609962 (±0.004199399)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 219.38399 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpened map

Fileemd_15895_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

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Additional map: Halfmap2 focused refinement half-dimer

Fileemd_15895_additional_2.map
AnnotationHalfmap2 focused refinement half-dimer
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AxesZYX

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Additional map: Halfmap1 focused refinement half-dimer

Fileemd_15895_additional_3.map
AnnotationHalfmap1 focused refinement half-dimer
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AxesZYX

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Additional map: Focused refinement half-dimer

Fileemd_15895_additional_4.map
AnnotationFocused refinement half-dimer
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AxesZYX

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Half map: #2

Fileemd_15895_half_map_1.map
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Half map: #1

Fileemd_15895_half_map_2.map
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Sample components

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Entire : Upright dimer KimA with c-di-AMP bound

EntireName: Upright dimer KimA with c-di-AMP bound
Components
  • Complex: Upright dimer KimA with c-di-AMP bound
    • 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

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

SupramoleculeName: Upright dimer KimA with c-di-AMP bound / 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

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.25 mg/mL
BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE
DetailsReconstituted in amphipols PMAL C8

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Electron microscopy

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

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

10092

Initial angle assignmentType: OTHER / Software - Name: RELION
Final angle assignmentType: OTHER / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 296000
FSC plot (resolution estimation)

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Atomic model buiding 1

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

PDB-8b71:
Upright KimA dimer with bound c-di-AMP from B. subtilis

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