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- EMDB-25981: SthK closed state, cAMP-bound in the presence of detergent -

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Basic information

Entry
Database: EMDB / ID: EMD-25981
TitleSthK closed state, cAMP-bound in the presence of detergent
Map dataCyclic nucleotide-gate ion channel SthK in detergent (DDM) in the presence of ligand (cAMP)
Sample
  • Complex: closed conformation of SthK bound to cAMP
    • Protein or peptide: Putative transcriptional regulator, Crp/Fnr family
  • Ligand: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
  • Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
Keywordscyclic nucleotide-gated channel / lipid modulation / pacemaker channel / TRANSPORT PROTEIN
Function / homology
Function and homology information


intracellular cyclic nucleotide activated cation channel complex / intracellularly cGMP-activated cation channel activity / intracellularly cAMP-activated cation channel activity / cGMP binding / protein-containing complex binding / plasma membrane
Similarity search - Function
Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Potassium channel domain / Ion channel / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold
Similarity search - Domain/homology
Transcriptional regulator, Crp/Fnr family
Similarity search - Component
Biological speciesSpirochaeta thermophila (strain ATCC 700085 / DSM 6578 / Z-1203) (bacteria) / Spirochaeta thermophila DSM 6578 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsRheinberger J / Schmidpeter PA
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM124451 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM088352 United States
American Heart Association18POST33960309 United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Anionic lipids unlock the gates of select ion channels in the pacemaker family.
Authors: Philipp A M Schmidpeter / Di Wu / Jan Rheinberger / Paul M Riegelhaupt / Haiping Tang / Carol V Robinson / Crina M Nimigean /
Abstract: Lipids play important roles in regulating membrane protein function, but the molecular mechanisms used are elusive. Here we investigated how anionic lipids modulate SthK, a bacterial pacemaker ...Lipids play important roles in regulating membrane protein function, but the molecular mechanisms used are elusive. Here we investigated how anionic lipids modulate SthK, a bacterial pacemaker channel homolog, and HCN2, whose activity contributes to pacemaking in the heart and brain. Using SthK allowed the reconstitution of purified channels in controlled lipid compositions for functional and structural assays that are not available for the eukaryotic channels. We identified anionic lipids bound tightly to SthK and their exact binding locations and determined that they potentiate channel activity. Cryo-EM structures in the most potentiating lipids revealed an open state and identified a nonannular lipid bound with its headgroup near an intersubunit salt bridge that clamps the intracellular channel gate shut. Breaking this conserved salt bridge abolished lipid modulation in SthK and eukaryotic HCN2 channels, indicating that anionic membrane lipids facilitate channel opening by destabilizing these interactions. Our findings underline the importance of state-dependent protein-lipid interactions.
History
DepositionJan 17, 2022-
Header (metadata) releaseOct 26, 2022-
Map releaseOct 26, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25981.png

Downloads & links

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Map

FileDownload / File: emd_25981.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCyclic nucleotide-gate ion channel SthK in detergent (DDM) in the presence of ligand (cAMP)
Voxel sizeX=Y=Z: 1.0961 Å
Density
Contour LevelBy AUTHOR: 0.022
Minimum - Maximum-0.07956422 - 0.15640497
Average (Standard dev.)0.000014761593 (±0.0042583533)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 280.6016 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25981_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_25981_msk_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Postprocessed map for SthK after refinement focused on...

Fileemd_25981_additional_1.map
AnnotationPostprocessed map for SthK after refinement focused on transmembrane domain
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 1 of the full length...

Fileemd_25981_half_map_1.map
AnnotationUnfiltered half map 1 of the full length SthK, used in refinement and gold standard FSC resolution calculation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 2 of the full length...

Fileemd_25981_half_map_2.map
AnnotationUnfiltered half map 2 of the full length SthK, used in refinement and gold standard FSC resolution calculation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : closed conformation of SthK bound to cAMP

EntireName: closed conformation of SthK bound to cAMP
Components
  • Complex: closed conformation of SthK bound to cAMP
    • Protein or peptide: Putative transcriptional regulator, Crp/Fnr family
  • Ligand: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
  • Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate

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Supramolecule #1: closed conformation of SthK bound to cAMP

SupramoleculeName: closed conformation of SthK bound to cAMP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: SthK was purified in dodecyl maltoside (DDM).
Source (natural)Organism: Spirochaeta thermophila (strain ATCC 700085 / DSM 6578 / Z-1203) (bacteria)

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Macromolecule #1: Putative transcriptional regulator, Crp/Fnr family

MacromoleculeName: Putative transcriptional regulator, Crp/Fnr family / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Spirochaeta thermophila DSM 6578 (bacteria) / Strain: ATCC 700085 / DSM 6578 / Z-1203
Molecular weightTheoretical: 51.118574 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAKDIGINSD PNSSSVDKLM KSSGVSNPTY TLVWKVWILA VTLYYAIRIP LTLVFPSLFS PLLPLDILAS LALIADIPLD LAFESRRTS GRKPTLLAPS RLPDLLAALP LDLLVFALHL PSPLSLLSLV RLLKLISVQR SATRILSYRI NPALLRLLSL V GFILLAAH ...String:
MAKDIGINSD PNSSSVDKLM KSSGVSNPTY TLVWKVWILA VTLYYAIRIP LTLVFPSLFS PLLPLDILAS LALIADIPLD LAFESRRTS GRKPTLLAPS RLPDLLAALP LDLLVFALHL PSPLSLLSLV RLLKLISVQR SATRILSYRI NPALLRLLSL V GFILLAAH GIACGWMSLQ PPSENPAGTR YLSAFYWTIT TLTTIGYGDI TPSTPTQTVY TIVIELLGAA MYGLVIGNIA SL VSKLDAA KLLHRERVER VTAFLSYKRI SPELQRRIIE YFDYLWETRR GYEEREVLKE LPHPLRLAVA MEIHGDVIEK VPL FKGAGE EFIRDIILHL EPVIYGPGEY IIRAGEMGSD VYFINRGSVE VLSADEKTRY AILSEGQFFG EMALILRAPR TATV RARAF CDLYRLDKET FDRILSRYPE IAAQIQELAV RRKELESSGL VPRGSVKHHH H

UniProtKB: Transcriptional regulator, Crp/Fnr family

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Macromolecule #2: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE

MacromoleculeName: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / type: ligand / ID: 2 / Number of copies: 4 / Formula: CMP
Molecular weightTheoretical: 329.206 Da
Chemical component information

ChemComp-CMP:
ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE

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Macromolecule #3: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]o...

MacromoleculeName: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
type: ligand / ID: 3 / Number of copies: 52 / Formula: PGW
Molecular weightTheoretical: 749.007 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

Concentration8 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
10.0 mMHEPES
100.0 mMpotassium chlorideKCl
2.0 mMcAMP
3.0 mMFos-Choline-8, Fluorinated
0.5 mMDDM
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV
DetailsWT SthK in dodecyl maltoside (DDM)

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: TFS Cs corrector / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 2561 / Average exposure time: 8.0 sec. / Average electron dose: 55.77 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 45616 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.001 mm / Nominal defocus max: 2.75 µm / Nominal defocus min: 1.25 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 365834
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5u6o


Details: map of the pdb model, lowpass filtered to 40A, was generated in chimara using 'molmap' command
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 30917
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6cju


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7tkt:
SthK closed state, cAMP-bound in the presence of detergent

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