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- EMDB-20650: Calcium-bound MthK gating ring state 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-20650
TitleCalcium-bound MthK gating ring state 2
Map dataCalcium-bound MthK gating ring state 2
Sample
  • Complex: Calcium-bound MthK gating ring
    • Protein or peptide: Calcium-gated potassium channel MthK
  • Ligand: CALCIUM IONCalcium
  • Ligand: water
KeywordsMthK / TRANSPORT PROTEIN
Function / homology
Function and homology information


monoatomic cation transmembrane transporter activity / potassium ion transport / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Regulator of K+ conductance, N-terminal / TrkA-N domain / RCK N-terminal domain profile. / Regulator of K+ conductance, C-terminal / Regulator of K+ conductance, C-terminal domain superfamily / TrkA-C domain / RCK C-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: 3.2 Å
AuthorsFan C / Nimigean C
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01GM088352 United States
CitationJournal: Nature / Year: 2020
Title: Ball-and-chain inactivation in a calcium-gated potassium channel.
Authors: Chen Fan / Nattakan Sukomon / Emelie Flood / Jan Rheinberger / Toby W Allen / Crina M Nimigean /
Abstract: Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present. In neurons, inactivation of both sodium and potassium channels is ...Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present. In neurons, inactivation of both sodium and potassium channels is crucial for the generation of action potentials and regulation of firing frequency. A cytoplasmic domain of either the channel or an accessory subunit is thought to plug the open pore to inactivate the channel via a 'ball-and-chain' mechanism. Here we use cryo-electron microscopy to identify the molecular gating mechanism in calcium-activated potassium channels by obtaining structures of the MthK channel from Methanobacterium thermoautotrophicum-a purely calcium-gated and inactivating channel-in a lipid environment. In the absence of Ca, we obtained a single structure in a closed state, which was shown by atomistic simulations to be highly flexible in lipid bilayers at ambient temperature, with large rocking motions of the gating ring and bending of pore-lining helices. In Ca-bound conditions, we obtained several structures, including multiple open-inactivated conformations, further indication of a highly dynamic protein. These different channel conformations are distinguished by rocking of the gating rings with respect to the transmembrane region, indicating symmetry breakage across the channel. Furthermore, in all conformations displaying open channel pores, the N terminus of one subunit of the channel tetramer sticks into the pore and plugs it, with free energy simulations showing that this is a strong interaction. Deletion of this N terminus leads to functionally non-inactivating channels and structures of open states without a pore plug, indicating that this previously unresolved N-terminal peptide is responsible for a ball-and-chain inactivation mechanism.
History
DepositionAug 28, 2019-
Header (metadata) releaseSep 11, 2019-
Map releaseMar 25, 2020-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6u5n
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20650.png

Downloads & links

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Map

FileDownload / File: emd_20650.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCalcium-bound MthK gating ring state 2
Voxel sizeX=Y=Z: 1.096 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.15968238 - 0.2749207
Average (Standard dev.)0.00008746376 (±0.005876196)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 263.03998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0961.0961.096
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z263.040263.040263.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.1600.2750.000

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Supplemental data

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Sample components

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Entire : Calcium-bound MthK gating ring

EntireName: Calcium-bound MthK gating ring
Components
  • Complex: Calcium-bound MthK gating ring
    • Protein or peptide: Calcium-gated potassium channel MthK
  • Ligand: CALCIUM IONCalcium
  • Ligand: water

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Supramolecule #1: Calcium-bound MthK gating ring

SupramoleculeName: Calcium-bound MthK gating ring / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Calcium bound MthK gating ring
Source (natural)Organism: Methanothermobacter thermautotrophicus (archaea)
Molecular weightTheoretical: 200 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: 37.35616 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVLVIEIIRK HLPRVLKVPA TRILLLVLAV IIYGTAGFHF IEGESWTVSL YWTFVTIATV GYGDYSPSTP LGMYFTVTLI VLGIGTFAV AVERLLEFLI NREQMKLMGL IDVAKSRHVV ICGWSESTLE CLRELRGSEV FVLAEDENVR KKVLRSGANF V HGDPTRVS ...String:
MVLVIEIIRK HLPRVLKVPA TRILLLVLAV IIYGTAGFHF IEGESWTVSL YWTFVTIATV GYGDYSPSTP LGMYFTVTLI VLGIGTFAV AVERLLEFLI 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: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 24 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 8 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

BufferpH: 8.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
100.0 mMpotassium chlorideKCl
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 296 K / Instrument: FEI VITROBOT MARK IV / Details: blotting for 2 seconds (blot force 0).

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -2.4 µm / Nominal defocus min: -1.4000000000000001 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 8.0 sec. / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3ldc

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 113103

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-6u5n:
Calcium-bound MthK gating ring state 2

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