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- EMDB-2527: The electron crystallography structure of the cAMP-free potassium... -

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Basic information

Entry
Database: EMDB / ID: EMD-2527
TitleThe electron crystallography structure of the cAMP-free potassium channel MloK1
Map dataMloK1 without cAMP
Sample
  • Sample: MloK1 without cAMP
  • Protein or peptide: MloK1
KeywordsElectron crystallography / 2dx / voltage gated potassium channel / CNBD / 2D crystal
Function / homology
Function and homology information


transport / intracellularly cyclic nucleotide-activated monoatomic cation channel activity / membrane => GO:0016020 / potassium channel activity / cAMP binding / monoatomic ion transport / potassium ion transmembrane transport / potassium ion transport / nucleotide binding / protein-containing complex binding ...transport / intracellularly cyclic nucleotide-activated monoatomic cation channel activity / membrane => GO:0016020 / potassium channel activity / cAMP binding / monoatomic ion transport / potassium ion transmembrane transport / potassium ion transport / nucleotide binding / protein-containing complex binding / identical protein binding / membrane / plasma membrane
Similarity search - Function
: / 1-aminocyclopropane-1-carboxylate deaminase/D-cysteine desulfhydrase / Cyclic nucleotide-regulated ion channel, N-terminal / : / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-binding, conserved site / Potassium channel domain / Cyclic nucleotide-monophosphate binding domain ...: / 1-aminocyclopropane-1-carboxylate deaminase/D-cysteine desulfhydrase / Cyclic nucleotide-regulated ion channel, N-terminal / : / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-binding, conserved site / Potassium channel domain / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold / RmlC-like jelly roll fold / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Cyclic nucleotide-gated potassium channel mll3241
Similarity search - Component
Biological speciesMesorhizobium loti (bacteria)
Methodelectron crystallography / cryo EM / Resolution: 7.0 Å
AuthorsKowal J / Chami M / Baumgartner P / Arheit M / Chiu P-L / Rangl M / Scheuring S / Schroeder GF / Nimigean CM / Stahlberg H
CitationJournal: Nat Commun / Year: 2014
Title: Ligand-induced structural changes in the cyclic nucleotide-modulated potassium channel MloK1.
Authors: Julia Kowal / Mohamed Chami / Paul Baumgartner / Marcel Arheit / Po-Lin Chiu / Martina Rangl / Simon Scheuring / Gunnar F Schröder / Crina M Nimigean / Henning Stahlberg /
Abstract: Cyclic nucleotide-modulated ion channels are important for signal transduction and pacemaking in eukaryotes. The molecular determinants of ligand gating in these channels are still unknown, mainly ...Cyclic nucleotide-modulated ion channels are important for signal transduction and pacemaking in eukaryotes. The molecular determinants of ligand gating in these channels are still unknown, mainly because of a lack of direct structural information. Here we report ligand-induced conformational changes in full-length MloK1, a cyclic nucleotide-modulated potassium channel from the bacterium Mesorhizobium loti, analysed by electron crystallography and atomic force microscopy. Upon cAMP binding, the cyclic nucleotide-binding domains move vertically towards the membrane, and directly contact the S1-S4 voltage sensor domains. This is accompanied by a significant shift and tilt of the voltage sensor domain helices. In both states, the inner pore-lining helices are in an 'open' conformation. We propose a mechanism in which ligand binding can favour pore opening via a direct interaction between the cyclic nucleotide-binding domains and voltage sensors. This offers a simple mechanistic hypothesis for the coupling between ligand gating and voltage sensing in eukaryotic HCN channels.
History
DepositionDec 4, 2013-
Header (metadata) releaseJan 8, 2014-
Map releaseJan 15, 2014-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4chw
  • Surface level: 2.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2527.png

Downloads & links

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Map

FileDownload / File: emd_2527.map.gz / Format: CCP4 / Size: 4.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMloK1 without cAMP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.98 Å/pix.
x 107 pix.
= 104.325 Å		0.98 Å/pix.
x 107 pix.
= 104.325 Å		0.98 Å/pix.
x 107 pix.
= 104.325 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.975 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.3 / Movie #1: 2.3
Minimum - Maximum-1.16991782 - 4.0808568
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin55133
Dimensions107107107
Spacing107107107
CellA=B=C: 104.325005 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9750.9750.975
M x/y/z107107107
origin x/y/z0.0000.0000.000
length x/y/z104.325104.325104.325
α/β/γ90.00090.00090.000
start NX/NY/NZ-300-300-299
NX/NY/NZ600600600
MAP C/R/S123
start NC/NR/NS55133
NC/NR/NS107107107
D min/max/mean-1.1704.081-0.000

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Supplemental data

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Sample components

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Entire : MloK1 without cAMP

EntireName: MloK1 without cAMP
Components
  • Sample: MloK1 without cAMP
  • Protein or peptide: MloK1

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Supramolecule #1000: MloK1 without cAMP

SupramoleculeName: MloK1 without cAMP / type: sample / ID: 1000 / Oligomeric state: tetramer / Number unique components: 1
Molecular weightTheoretical: 148 KDa
Method: Calculated from sequence for the tetrameric assembly. Lipids are not included.

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Macromolecule #1: MloK1

MacromoleculeName: MloK1 / type: protein_or_peptide / ID: 1 / Name.synonym: MlotiK1 / Details: cAMP not present in buffer / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: Yes
Source (natural)Organism: Mesorhizobium loti (bacteria) / synonym: Rhizobium / Location in cell: Membrane
Molecular weightTheoretical: 148 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pASK90
SequenceUniProtKB: Cyclic nucleotide-gated potassium channel mll3241
GO: transport, monoatomic ion transport, potassium ion transport, potassium ion transmembrane transport, nucleotide binding, potassium channel activity, cAMP binding, plasma membrane, membrane, membrane => GO:0016020
InterPro: Potassium channel domain, Cyclic nucleotide-binding domain superfamily, Cyclic nucleotide-binding, conserved site, Cyclic nucleotide-binding domain, INTERPRO: IPR003091, 1-aminocyclopropane- ...InterPro: Potassium channel domain, Cyclic nucleotide-binding domain superfamily, Cyclic nucleotide-binding, conserved site, Cyclic nucleotide-binding domain, INTERPRO: IPR003091, 1-aminocyclopropane-1-carboxylate deaminase/D-cysteine desulfhydrase, RmlC-like jelly roll fold

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

Concentration0.7 mg/mL
BufferpH: 7.6 / Details: 20 mM KCl, 1 mM BaCl2, 1 mM EDTA, 20 mM Tris
GridDetails: Holey carbon film (Quantifoil) covered with ultra thin carbon film. Vitrified in crystallization buffer solution by plunge freezing into ethane.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 3 seconds before plunging
DetailsDialysis
Crystal formationDetails: Dialysis

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
TemperatureMin: 85 K / Average: 85 K
DateMar 1, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 5 µm / Number real images: 78 / Average electron dose: 5 e/Å2 / Od range: 1.4 / Bits/pixel: 16
Tilt angle min0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.077 µm / Nominal defocus min: 0.655 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt angle max: 46 / Tilt series - Axis1 - Min angle: 0 ° / Tilt series - Axis1 - Max angle: 46 °

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Image processing

Details2dx
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: OTHER / Software - Name: 2dx / Details: Resolution was limited to 7 x 7 x 12 Angstroems
Crystal parametersUnit cell - A: 130 Å / Unit cell - B: 130 Å / Unit cell - C: 400 Å / Unit cell - γ: 90.0 ° / Unit cell - α: 90.0 ° / Unit cell - β: 90.0 ° / Plane group: P 4 21 2

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