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- EMDB-29605: TpeA bound closed MthK-A88F mutant in nanodisc -

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Basic information

Entry
Database: EMDB / ID: EMD-29605
TitleTpeA bound closed MthK-A88F mutant in nanodisc
Map data
Sample
  • Complex: TpeA bound MthK A88F mutant in 0 Ca2+
    • Protein or peptide: Calcium-gated potassium channel MthK
  • Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
  • Ligand: 1-(tripentyl-$l^{4}-azanyl)pentane
  • Ligand: POTASSIUM IONPotassium
KeywordsMTHK / TRANSPORT PROTEIN / ION CHANNEL / Blocker TpeA / MEMBRANE 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: 2.88 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: 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
DepositionJan 27, 2023-
Header (metadata) releaseOct 11, 2023-
Map releaseOct 11, 2023-
UpdateJan 3, 2024-
Current statusJan 3, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29605.png

Downloads & links

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Map

FileDownload / File: emd_29605.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.0826 Å
Density
Contour LevelBy AUTHOR: 0.0178
Minimum - Maximum-0.038262926 - 0.11003029
Average (Standard dev.)0.00028924318 (±0.0031955582)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 138.5728 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29605_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_29605_additional_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29605_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29605_half_map_2.map
Projections & Slices
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Sample components

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Entire : TpeA bound MthK A88F mutant in 0 Ca2+

EntireName: TpeA bound MthK A88F mutant in 0 Ca2+
Components
  • Complex: TpeA bound MthK A88F mutant in 0 Ca2+
    • Protein or peptide: Calcium-gated potassium channel MthK
  • Ligand: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
  • Ligand: 1-(tripentyl-$l^{4}-azanyl)pentane
  • Ligand: POTASSIUM IONPotassium

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Supramolecule #1: TpeA bound MthK A88F mutant in 0 Ca2+

SupramoleculeName: TpeA bound MthK A88F 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: 37.432258 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVLVIEIIRK HLPRVLKVPA TRILLLVLAV IIYGTAGFHF IEGESWTVSL YWTFVTIATV GYGDYSPSTP LGMYFTVTLI VLGIGTFFV AVERLLEFLI NREQMKLMGL IDVAKSRHVV ICGWSESTLE CLRELRGSEV FVLAEDENVR KKVLRSGANF V HGDPTRVS ...String:
MVLVIEIIRK HLPRVLKVPA TRILLLVLAV IIYGTAGFHF IEGESWTVSL YWTFVTIATV GYGDYSPSTP LGMYFTVTLI VLGIGTFFV 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: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]o...

MacromoleculeName: (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate
type: ligand / ID: 2 / Number of copies: 4 / Formula: PGW
Molecular weightTheoretical: 749.007 Da

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Macromolecule #3: 1-(tripentyl-$l^{4}-azanyl)pentane

MacromoleculeName: 1-(tripentyl-$l^{4}-azanyl)pentane / type: ligand / ID: 3 / Number of copies: 1 / Formula: YQ1
Molecular weightTheoretical: 298.57 Da
Chemical component information

ChemComp-YQ1:
1-(tripentyl-$l^{4}-azanyl)pentane

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Macromolecule #4: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 4 / Number of copies: 2 / 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 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: 3.0 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 58.9 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:

6u6d

Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 4)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 4)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Number images used: 105404
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-8fz7:
TpeA bound closed MthK-A88F mutant in nanodisc

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