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- PDB-6cjt: Structure of the SthK cyclic nucleotide-gated potassium channel i... -

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Basic information

Entry
Database: PDB / ID: 6cjt
TitleStructure of the SthK cyclic nucleotide-gated potassium channel in complex with cGMP
ComponentsSthK cyclic nucleotide-gated potassium channel
KeywordsTRANSPORT PROTEIN / ion channel / tetramer / lipid / cGMP
Function / homologyCyclic nucleotide-binding domain / Potassium channel domain / RmlC-like jelly roll fold / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-binding-like / Cyclic nucleotide-binding domain / Ion channel / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding domain signature 2. / cAMP/cGMP binding motif profile. ...Cyclic nucleotide-binding domain / Potassium channel domain / RmlC-like jelly roll fold / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-binding-like / Cyclic nucleotide-binding domain / Ion channel / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding domain signature 2. / cAMP/cGMP binding motif profile. / integral component of membrane / Putative transcriptional regulator, Crp/Fnr family
Function and homology information
Specimen sourceSpirochaeta thermophila (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.46 Å resolution
AuthorsNimigean, C.M. / Rheinberger, J.
CitationJournal: Elife / Year: 2018
Title: Ligand discrimination and gating in cyclic nucleotide-gated ion channels from apo and partial agonist-bound cryo-EM structures.
Authors: Jan Rheinberger / Xiaolong Gao / Philipp Am Schmidpeter / Crina M Nimigean
Abstract: Cyclic nucleotide-modulated channels have important roles in visual signal transduction and pacemaking. Binding of cyclic nucleotides (cAMP/cGMP) elicits diverse functional responses in different ...Cyclic nucleotide-modulated channels have important roles in visual signal transduction and pacemaking. Binding of cyclic nucleotides (cAMP/cGMP) elicits diverse functional responses in different channels within the family despite their high sequence and structure homology. The molecular mechanisms responsible for ligand discrimination and gating are unknown due to lack of correspondence between structural information and functional states. Using single particle cryo-electron microscopy and single-channel recording, we assigned functional states to high-resolution structures of SthK, a prokaryotic cyclic nucleotide-gated channel. The structures for apo, cAMP-bound, and cGMP-bound SthK in lipid nanodiscs, correspond to no, moderate, and low single-channel activity, respectively, consistent with the observation that all structures are in resting, closed states. The similarity between apo and ligand-bound structures indicates that ligand-binding domains are strongly coupled to pore and SthK gates in an allosteric, concerted fashion. The different orientations of cAMP and cGMP in the 'resting' and 'activated' structures suggest a mechanism for ligand discrimination.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 26, 2018 / Release: Aug 1, 2018

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Structure visualization

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Assembly

Deposited unit
A: SthK cyclic nucleotide-gated potassium channel
B: SthK cyclic nucleotide-gated potassium channel
C: SthK cyclic nucleotide-gated potassium channel
D: SthK cyclic nucleotide-gated potassium channel
hetero molecules


Theoretical massNumber of molelcules
Total (without water)223,83132
Polyers204,4744
Non-polymers19,35728
Water0
1


  • idetical with deposited unit
  • defined by author
  • Evidence: cross-linking, gel filtration microscopy
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)38390
ΔGint (kcal/M)-253
Surface area (Å2)76510

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Components

#1: Protein/peptide
SthK cyclic nucleotide-gated potassium channel


Mass: 51118.574 Da / Num. of mol.: 4 / Source: (gene. exp.) Spirochaeta thermophila (bacteria) / Strain: ATCC 700085 / DSM 6578 / Z-1203 / Gene: Spith_0644 / Production host: Escherichia coli (E. coli) / References: UniProt: G0GA88
#2: Chemical
ChemComp-PCG / CYCLIC GUANOSINE MONOPHOSPHATE


Mass: 345.205 Da / Num. of mol.: 4 / Formula: C10H12N5O7P / Cyclic guanosine monophosphate
#3: Chemical...
ChemComp-PGW / (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate / 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-(1-glycerol)] / PHOSPHATIDYLGLYCEROL


Mass: 749.007 Da / Num. of mol.: 24 / Formula: C40H77O10P / Phosphatidylglycerol / Comment: phospholipid *YM

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: SthK cyclic nucleotide-gated potassium channel / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Spirochaeta thermophila DSM 6578 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C4
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 91800 / Symmetry type: POINT
Least-squares processHighest resolution: 3.46 Å

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