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- EMDB-2709: E. coli potassium channel 3D structure by electron crystallography -

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Basic information

Entry
Database: EMDB / ID: EMD-2709
TitleE. coli potassium channel 3D structure by electron crystallography
Map data2D crystals of KchTM, which contains the transmembrane part of Kch, a putative potassium channel from Escherichia coli. The structure of MlotiK1 (PDB ID: 3BEH) was docked as a similar protein to Kch into the overall 3D map using Chimera
Sample
  • Sample: voltage-gated potassium channel, overall map covering the whole unit cell
  • Protein or peptide: Kch
KeywordsPotassium channel / 6TM / RCK
Function / homology
Function and homology information


transport / membrane => GO:0016020 / potassium channel activity / monoatomic ion transport / potassium ion transmembrane transport / potassium ion transport / protein-containing complex / identical protein binding / membrane / plasma membrane
Similarity search - Function
: / : / TrkA-N domain / Regulator of K+ conductance, N-terminal / Regulator of K+ conductance, N-terminal / RCK N-terminal domain profile. / Voltage-gated potassium channel / Potassium channel domain / Ion channel / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Voltage-gated potassium channel Kch
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodelectron crystallography / cryo EM / negative staining / Resolution: 10.0 Å
AuthorsKuang Q / Purhonen P / Jegerschold C / Koeck PJB / Hebert H
CitationJournal: Structure / Year: 2015
Title: Free RCK arrangement in Kch, a putative escherichia coli potassium channel, as suggested by electron crystallography.
Authors: Qie Kuang / Pasi Purhonen / Caroline Jegerschöld / Philip J B Koeck / Hans Hebert /
Abstract: The ligand-gated potassium channels are stimulated by various kinds of messengers. Previous studies showed that ligand-gated potassium channels containing RCK domains (the regulator of the ...The ligand-gated potassium channels are stimulated by various kinds of messengers. Previous studies showed that ligand-gated potassium channels containing RCK domains (the regulator of the conductance of potassium ion) form a dimer of tetramer structure through the RCK octameric gating ring in the presence of detergent. Here, we have analyzed the structure of Kch, a channel of this type from Escherichia coli, in a lipid environment using electron crystallography. By combining information from the 3D map of the transmembrane part of the protein and docking of an atomic model of a potassium channel, we conclude that the RCK domains face the solution and that an RCK octameric gating ring arrangement does not form under our crystallization condition. Our findings may be applied to other potassium channels that have an RCK gating ring arrangement.
History
DepositionJul 11, 2014-
Header (metadata) releaseJul 23, 2014-
Map releaseJan 21, 2015-
UpdateJan 21, 2015-
Current statusJan 21, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0156
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0156
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2709.jpg

Downloads & links

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Map

FileDownload / File: emd_2709.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation2D crystals of KchTM, which contains the transmembrane part of Kch, a putative potassium channel from Escherichia coli. The structure of MlotiK1 (PDB ID: 3BEH) was docked as a similar protein to Kch into the overall 3D map using Chimera
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesY (Sec.)X (Row.)Z (Col.)
3.17 Å/pix.
x 48 pix.
= 203.078 Å		3.25 Å/pix.
x 88 pix.
= 156. Å		3.13 Å/pix.
x 64 pix.
= 275. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 3.25 Å / Y: 3.1731 Å / Z: 3.125 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0156 / Movie #1: 0.0156
Minimum - Maximum-0.03797268 - 0.06396198
Average (Standard dev.)-0.00009359 (±0.01079917)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZXY
Origin-22-32-12
Dimensions886448
Spacing644888
CellA: 156.0 Å / B: 203.0784 Å / C: 275.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.253.173093753.125
M x/y/z486488
origin x/y/z0.0000.0000.000
length x/y/z156.000203.078275.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-22-12-32
NX/NY/NZ884864
MAP C/R/S312
start NC/NR/NS-32-22-12
NC/NR/NS648848
D min/max/mean-0.0380.064-0.000

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Supplemental data

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Sample components

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Entire : voltage-gated potassium channel, overall map covering the whole u...

EntireName: voltage-gated potassium channel, overall map covering the whole unit cell
Components
  • Sample: voltage-gated potassium channel, overall map covering the whole unit cell
  • Protein or peptide: Kch

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Supramolecule #1000: voltage-gated potassium channel, overall map covering the whole u...

SupramoleculeName: voltage-gated potassium channel, overall map covering the whole unit cell
type: sample / ID: 1000 / Oligomeric state: tetramers / Number unique components: 1

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Macromolecule #1: Kch

MacromoleculeName: Kch / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Oligomeric state: tetramer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12 / Location in cell: inner membrane
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: c43 / Recombinant plasmid: pSP19T7LT
SequenceUniProtKB: Voltage-gated potassium channel Kch
GO: transport, monoatomic ion transport, potassium ion transport, plasma membrane, potassium ion transmembrane transport, membrane, membrane => GO:0016020
InterPro: INTERPRO: IPR003091, Regulator of K+ conductance, N-terminal, Voltage-gated potassium channel

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

Concentration0.7 mg/mL
BufferDetails: 25 mM Na-acetate at pH 7, 20% glycerol, 100 mM KCl, 0.1 mM EDTA, 1 mM reduced glutathione, 50 mM MgCl2, with or without 5% (w/v) glycine
StainingType: NEGATIVE / Details: Embedded in trehalose
GridDetails: Copper grid with carbon
VitrificationCryogen name: NITROGEN / Chamber humidity: 20 % / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER / Details: Manual freezing
DetailsCrystal grown by dialysis away the detergent
Crystal formationDetails: Crystal grown by dialysis away the detergent

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Electron microscopy

MicroscopeJEOL 2100F
TemperatureAverage: 95 K
DateMar 1, 2010
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 33 / Average electron dose: 12 e/Å2 / 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 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt angle max: 45 / Tilt series - Axis1 - Min angle: 0 ° / Tilt series - Axis1 - Max angle: 45 °

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Image processing

DetailsThe 2dx software suite was used to process each individual image to extract its amplitude and phase information. The MRC image processing package was used to merge the images.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: OTHER / Software - Name: MRC
Crystal parametersUnit cell - A: 143 Å / Unit cell - B: 82 Å / Unit cell - C: 200 Å / Unit cell - γ: 90 ° / Plane group: C 1 2
CTF correctionDetails: Each image

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Atomic model buiding 1

Initial modelPDB ID:

3beh

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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