+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-24670 | |||||||||
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Title | SthK Y26F Closed State | |||||||||
Map data | ||||||||||
Sample |
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Keywords | Cyclic nucleotide-gated ion channel / TRANSPORT PROTEIN | |||||||||
Biological species | Spirochaeta thermophila (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.0 Å | |||||||||
Authors | Gao X / Nimigean C | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Nat Commun / Year: 2022 Title: Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel. Authors: Xiaolong Gao / Philipp A M Schmidpeter / Vladimir Berka / Ryan J Durham / Chen Fan / Vasanthi Jayaraman / Crina M Nimigean / Abstract: Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel ...Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the intracellular gate, pointing to an inhibitory VSD at rest. We propose that in wild-type SthK, depolarization leads to such VSD displacements resulting in release of inhibition. In summary, we report conformational transitions along the activation pathway that reveal allosteric couplings between key sites integrating to open the intracellular gate. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_24670.map.gz | 4.5 MB | EMDB map data format | |
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Header (meta data) | emd-24670-v30.xml emd-24670.xml | 13.6 KB 13.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_24670_fsc.xml | 8.6 KB | Display | FSC data file |
Images | emd_24670.png | 153.3 KB | ||
Filedesc metadata | emd-24670.cif.gz | 6.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-24670 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-24670 | HTTPS FTP |
-Validation report
Summary document | emd_24670_validation.pdf.gz | 410.9 KB | Display | EMDB validaton report |
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Full document | emd_24670_full_validation.pdf.gz | 410.5 KB | Display | |
Data in XML | emd_24670_validation.xml.gz | 10.4 KB | Display | |
Data in CIF | emd_24670_validation.cif.gz | 13.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-24670 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-24670 | HTTPS FTP |
-Related structure data
Related structure data | 7rshMC 7rtfC 7rtjC 7ru0C 7ryrC 7rysC M: atomic model generated by this map C: citing same article (ref.) |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_24670.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.05878 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
-Entire : SthK
Entire | Name: SthK |
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Components |
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-Supramolecule #1: SthK
Supramolecule | Name: SthK / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Y26F mutant of wild type SthK |
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Source (natural) | Organism: Spirochaeta thermophila (bacteria) |
-Macromolecule #1: SthK
Macromolecule | Name: SthK / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: Spirochaeta thermophila (bacteria) |
Molecular weight | Theoretical: 51.102574 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MAKDIGINSD PNSSSVDKLM KSSGVSNPTY TLVWKVWILA VTLYFAIRIP LTLVFPSLFS PLLPLDILAS LALIADIPLD LAFESRRTS GRKPTLLAPS RLPDLLAALP LDLLVFALHL PSPLSLLSLV RLLKLISVQR SATRILSYRI NPALLRLLSL V GFILLAAH ...String: MAKDIGINSD PNSSSVDKLM KSSGVSNPTY TLVWKVWILA VTLYFAIRIP 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 |
-Macromolecule #2: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
Macromolecule | Name: ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / type: ligand / ID: 2 / Number of copies: 4 / Formula: CMP |
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Molecular weight | Theoretical: 329.206 Da |
Chemical component information | ChemComp-CMP: |
-Macromolecule #3: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]o...
Macromolecule | Name: (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: 56 / Formula: PGW |
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Molecular weight | Theoretical: 749.007 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 7.5 mg/mL | |||||||||
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Buffer | pH: 7 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec. | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV | |||||||||
Details | Protein in nanodisc of DOPC/POPG/Cardiolipin |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 1.365 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |