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- PDB-8djb: MthK-A90L mutant in closed state with 0 Ca2+ -

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Basic information

Entry
Database: PDB / ID: 8djb
TitleMthK-A90L mutant in closed state with 0 Ca2+
ComponentsCalcium-gated potassium channel MthK
KeywordsION CHANNEL / MTHK / TRANSPORT PROTEIN
Function / homology
Function and homology information


monoatomic cation transmembrane transporter activity / potassium ion transport / metal ion binding / identical protein binding / plasma membrane
Similarity search - Function
: / TrkA-N domain / Regulator of K+ conductance, C-terminal / Regulator of K+ conductance, C-terminal domain superfamily / TrkA-C domain / RCK C-terminal domain profile. / Regulator of K+ conductance, N-terminal / RCK N-terminal domain profile. / Potassium channel domain / Ion channel / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
: / Calcium-gated potassium channel MthK
Similarity search - Component
Biological speciesMethanothermobacter thermautotrophicus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsAgarwal, S. / Nimigean, C.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH GM088352 United States
CitationJournal: Nat Chem Biol / Year: 2024
Title: Calcium-gated potassium channel blockade via membrane-facing fenestrations.
Authors: Chen Fan / Emelie Flood / Nattakan Sukomon / Shubhangi Agarwal / Toby W Allen / Crina M Nimigean /
Abstract: Quaternary ammonium blockers were previously shown to bind in the pore to block both open and closed conformations of large-conductance calcium-activated potassium (BK and MthK) channels. Because ...Quaternary ammonium blockers were previously shown to bind in the pore to block both open and closed conformations of large-conductance calcium-activated potassium (BK and MthK) channels. Because blocker entry was assumed through the intracellular entryway (bundle crossing), closed-pore access suggested that the gate was not at the bundle crossing. Structures of closed MthK, a Methanobacterium thermoautotrophicum homolog of BK channels, revealed a tightly constricted intracellular gate, leading us to investigate the membrane-facing fenestrations as alternative pathways for blocker access directly from the membrane. Atomistic free energy simulations showed that intracellular blockers indeed access the pore through the fenestrations, and a mutant channel with narrower fenestrations displayed no closed-state TPeA block at concentrations that blocked the wild-type channel. Apo BK channels display similar fenestrations, suggesting that blockers may use them as access paths into closed channels. Thus, membrane fenestrations represent a non-canonical pathway for selective targeting of specific channel conformations, opening novel ways to selectively drug BK channels.
History
DepositionJun 30, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 5, 2023Provider: repository / Type: Initial release
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Revision 1.1Oct 11, 2023Group: Data collection / Database references / Refinement description
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Calcium-gated potassium channel MthK
B: Calcium-gated potassium channel MthK
D: Calcium-gated potassium channel MthK
F: Calcium-gated potassium channel MthK
H: Calcium-gated potassium channel MthK
G: Calcium-gated potassium channel MthK
C: Calcium-gated potassium channel MthK
E: Calcium-gated potassium channel MthK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)299,2259
Polymers299,1868
Non-polymers391
Water00
1


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

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Components

#1: Protein
Calcium-gated potassium channel MthK


Mass: 37398.242 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanothermobacter thermautotrophicus (archaea)
Gene: mthK, MTH_1520 / Production host: Escherichia coli (E. coli) / References: UniProt: O27564
#2: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K
Has ligand of interestY
Has protein modificationN

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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: MthK A90L mutant in 0 Ca2+ / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: YES
Source (natural)Organism: Methanothermobacter thermautotrophicus (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.5
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Details: BLOTTING FOR 2 S (BLOT FORCE 1)

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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 defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 65.19 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
9PHENIXmodel refinement
10RELION4initial Euler assignment
11RELION4final Euler assignment
13RELION43D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80904 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6U6D

6u6d


Accession code: 6U6D / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.18 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00217020
ELECTRON MICROSCOPYf_angle_d0.37923024
ELECTRON MICROSCOPYf_dihedral_angle_d10.3546392
ELECTRON MICROSCOPYf_chiral_restr0.0432692
ELECTRON MICROSCOPYf_plane_restr0.0032988

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