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- EMDB-27459: MthK-A90L mutant in closed state with 0 Ca2+ -

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Basic information

Entry
Database: EMDB / ID: EMD-27459
TitleMthK-A90L mutant in closed state with 0 Ca2+
Map data
Sample
  • Complex: MthK A90L mutant in 0 Ca2+
    • Protein or peptide: Calcium-gated potassium channel MthK
  • Ligand: POTASSIUM ION
KeywordsMTHK / TRANSPORT PROTEIN / ION CHANNEL
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsAgarwal S / Nimigean CM
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseJul 5, 2023-
Map releaseJul 5, 2023-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27459.png

Downloads & links

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Map

FileDownload / File: emd_27459.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 300 pix.
= 324.9 Å		1.08 Å/pix.
x 300 pix.
= 324.9 Å		1.08 Å/pix.
x 300 pix.
= 324.9 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.083 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.018
Minimum - Maximum-0.1179212 - 0.19022892
Average (Standard dev.)0.000033822816 (±0.0036962535)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 324.9 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_27459_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27459_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MthK A90L mutant in 0 Ca2+

EntireName: MthK A90L mutant in 0 Ca2+
Components
  • Complex: MthK A90L mutant in 0 Ca2+
    • Protein or peptide: Calcium-gated potassium channel MthK
  • Ligand: POTASSIUM ION

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Supramolecule #1: MthK A90L mutant in 0 Ca2+

SupramoleculeName: MthK A90L mutant in 0 Ca2+ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Methanothermobacter thermautotrophicus (archaea)
Molecular weightTheoretical: 220 KDa

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Macromolecule #1: Calcium-gated potassium channel MthK

MacromoleculeName: Calcium-gated potassium channel MthK / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Methanothermobacter thermautotrophicus (archaea)
Molecular weightTheoretical: 37.398242 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVLVIEIIRK HLPRVLKVPA TRILLLVLAV IIYGTAGFHF IEGESWTVSL YWTFVTIATV GYGDYSPSTP LGMYFTVTLI VLGIGTFAV LVERLLEFLI NREQMKLMGL IDVAKSRHVV ICGWSESTLE CLRELRGSEV FVLAEDENVR KKVLRSGANF V HGDPTRVS ...String:
MVLVIEIIRK HLPRVLKVPA TRILLLVLAV IIYGTAGFHF IEGESWTVSL YWTFVTIATV GYGDYSPSTP LGMYFTVTLI VLGIGTFAV LVERLLEFLI NREQMKLMGL IDVAKSRHVV ICGWSESTLE CLRELRGSEV FVLAEDENVR KKVLRSGANF V HGDPTRVS DLEKANVRGA RAVIVDLESD SETIHCILGI RKIDESVRII AEAERYENIE QLRMAGADQV ISPFVISGRL MS RSIDDGY EAMFVQDVLA EESTRRMVEV PIPEGSKLEG VSVLDADIHD VTGVIIIGVG RGDELIIDPP RDYSFRAGDI ILG IGKPEE IERLKNYISA

UniProtKB: Calcium-gated potassium channel MthK

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Macromolecule #2: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: K
Molecular weightTheoretical: 39.098 Da

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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

Concentration7 mg/mL
BufferpH: 8.5
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Details: BLOTTING FOR 2 S (BLOT FORCE 1).

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 65.19 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING ONLY
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6u6d

Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number images used: 80904
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 4)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6u6d


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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