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- EMDB-25917: SthK open state, cAMP-bound in the presence of POPA -

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Basic information

Entry
Database: EMDB / ID: EMD-25917
TitleSthK open state, cAMP-bound in the presence of POPA
Map datafinal map from refinement, unsharpened
Sample
  • Complex: open conformation of SthK bound to cAMP
    • Protein or peptide: Putative transcriptional regulator, Crp/Fnr family
  • Ligand: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
  • Ligand: (2R)-1-(hexadecanoyloxy)-3-(phosphonooxy)propan-2-yl (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: 2.9 Å
AuthorsSchmidpeter PA / Nimigean CM
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 14, 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_25917.png

Downloads & links

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Map

FileDownload / File: emd_25917.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfinal map from refinement, unsharpened
Voxel sizeX=Y=Z: 1.278 Å
Density
Contour LevelBy AUTHOR: 0.0142
Minimum - Maximum-0.020735636 - 0.06995014
Average (Standard dev.)0.00000030737212 (±0.0017997392)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 327.168 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25917_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2, unfiltered

Fileemd_25917_half_map_1.map
Annotationhalf map 2, unfiltered
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1, unfiltered

Fileemd_25917_half_map_2.map
Annotationhalf map 1, unfiltered
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : open conformation of SthK bound to cAMP

EntireName: open conformation of SthK bound to cAMP
Components
  • Complex: open conformation of SthK bound to cAMP
    • Protein or peptide: Putative transcriptional regulator, Crp/Fnr family
  • Ligand: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
  • Ligand: (2R)-1-(hexadecanoyloxy)-3-(phosphonooxy)propan-2-yl (9Z)-octadec-9-enoate

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Supramolecule #1: open conformation of SthK bound to cAMP

SupramoleculeName: open conformation of SthK bound to cAMP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: SthK was reconstituted into nanodiscs containing POPA lipids.
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: (2R)-1-(hexadecanoyloxy)-3-(phosphonooxy)propan-2-yl (9Z)-octadec...

MacromoleculeName: (2R)-1-(hexadecanoyloxy)-3-(phosphonooxy)propan-2-yl (9Z)-octadec-9-enoate
type: ligand / ID: 3 / Number of copies: 24 / Formula: D21
Molecular weightTheoretical: 674.929 Da
Chemical component information

ChemComp-D21:
(2R)-1-(hexadecanoyloxy)-3-(phosphonooxy)propan-2-yl (9Z)-octadec-9-enoate

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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
3.0 mMcAMP
3.0 mMFos-Choline-8, Fluorinated
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298.15 K / Instrument: FEI VITROBOT MARK IV
DetailsWT SthK in MSP1E3 nanodiscs composed of 3:1 DOPC:POPA

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 5684 / Average exposure time: 2.6 sec. / Average electron dose: 50.812 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 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: 1561870
Startup modelType of model: OTHER / Details: initial model generated in Relion
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.3) / Number images used: 202148
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1.3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6vy0


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

PDB-7tj6:
SthK open state, cAMP-bound in the presence of POPA

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