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- PDB-7ru0: SthK R120A Open State 1 -

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Basic information

Entry
Database: PDB / ID: 7ru0
TitleSthK R120A Open State 1
ComponentsSthK
KeywordsTRANSPORT PROTEIN / Cyclic nucleotide-gated ion channel
Function / homologyADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
Function and homology information
Biological speciesSpirochaeta thermophila (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsGao, X. / Nimigean, C. / Schmidpeter, P.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: 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
DepositionAug 16, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 23, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 30, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: SthK
B: SthK
C: SthK
D: SthK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,4478
Polymers204,1304
Non-polymers1,3174
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
SthK


Mass: 51032.461 Da / Num. of mol.: 4 / Mutation: R120A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spirochaeta thermophila (bacteria) / Production host: Escherichia coli (E. coli)
#2: Chemical
ChemComp-CMP / ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / CYCLIC AMP / CAMP


Mass: 329.206 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H12N5O6P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: SthK / Type: COMPLEX / Details: R120A mutant of wild type SthK / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Spirochaeta thermophila (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2100 mMpotassium chlorideKCl1
SpecimenConc.: 7.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Protein in nanodisc of DOPC/POPG/Cardiolipin
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.459 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
2Leginonimage acquisition
4RELION3.1_BetaCTF correction
7PHENIXmodel fitting
9RELION3.1_Betainitial Euler assignment
10RELION3.1_Betafinal Euler assignment
11RELION3.1_Betaclassification
12RELION3.1_Beta3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3004702
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50037 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412568
ELECTRON MICROSCOPYf_angle_d1.12517140
ELECTRON MICROSCOPYf_dihedral_angle_d12.2121752
ELECTRON MICROSCOPYf_chiral_restr0.0572052
ELECTRON MICROSCOPYf_plane_restr0.0072100

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