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- EMDB-3907: The electron crystallography structure of the cAMP-bound potassiu... -

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Basic information

Entry
Database: EMDB / ID: EMD-3907
TitleThe electron crystallography structure of the cAMP-bound potassium channel MloK1 (PCO-refined)
Map dataPCO (Projective ConstraintOptimization) - refined 3D volume of MloK1 with cAMP
Sample
  • Complex: MloK1 tetramer
    • Protein or peptide: Cyclic nucleotide-gated potassium channel mll3241
  • Ligand: POTASSIUM IONPotassium
Function / homology
Function and homology information


potassium channel activity / cAMP binding / identical protein binding / plasma membrane
Similarity search - Function
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-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold ...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-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / 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) / Mesorhizobium loti MAFF303099 (bacteria)
Methodelectron crystallography / cryo EM / Resolution: 4.5 Å
AuthorsKowal J / Biyani N / Chami M / Scherer S / Rzepiela A / Baumgartner P / Upadhyay V / Nimigean C / Stahlberg H
CitationJournal: Structure / Year: 2018
Title: High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer.
Authors: Julia Kowal / Nikhil Biyani / Mohamed Chami / Sebastian Scherer / Andrzej J Rzepiela / Paul Baumgartner / Vikrant Upadhyay / Crina M Nimigean / Henning Stahlberg /
Abstract: Eukaryotic cyclic nucleotide-modulated channels perform their diverse physiological roles by opening and closing their pores to ions in response to cyclic nucleotide binding. We here present a ...Eukaryotic cyclic nucleotide-modulated channels perform their diverse physiological roles by opening and closing their pores to ions in response to cyclic nucleotide binding. We here present a structural model for the cyclic nucleotide-modulated potassium channel homolog from Mesorhizobium loti, MloK1, determined from 2D crystals in the presence of lipids. Even though crystals diffract electrons to only ∼10 Å, using cryoelectron microscopy (cryo-EM) and recently developed computational methods, we have determined a 3D map of full-length MloK1 in the presence of cyclic AMP (cAMP) at ∼4.5 Å isotropic 3D resolution. The structure provides a clear picture of the arrangement of the cyclic nucleotide-binding domains with respect to both the pore and the putative voltage sensor domains when cAMP is bound, and reveals a potential gating mechanism in the context of the lipid-embedded channel.
History
DepositionOct 8, 2017-
Header (metadata) releaseDec 20, 2017-
Map releaseDec 27, 2017-
UpdateJan 10, 2018-
Current statusJan 10, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 600
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 600
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6eo1
  • Surface level: 600
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3907_1.png

emd_3907_2.png

Downloads & links

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Map

FileDownload / File: emd_3907.map.gz / Format: CCP4 / Size: 9.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPCO (Projective ConstraintOptimization) - refined 3D volume of MloK1 with cAMP
Voxel sizeX: 0.955 Å / Y: 0.955 Å / Z: 1.045 Å
Density
Contour LevelBy AUTHOR: 600. / Movie #1: 600
Minimum - Maximum-499.997529999999983 - 2773.677000000000135
Average (Standard dev.)9.9917555 (±257.078700000000026)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin550
Dimensions112112201
Spacing112112201
CellA: 106.96 Å / B: 106.96 Å / C: 210.045 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9550.9551.045
M x/y/z112112201
origin x/y/z0.0000.0000.000
length x/y/z106.960106.960210.045
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS550
NC/NR/NS112112201
D min/max/mean-499.9982773.6779.992

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Supplemental data

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Additional map: Back-projected 3D volume of MloK1 with cAMP

Fileemd_3907_additional.map
AnnotationBack-projected 3D volume of MloK1 with cAMP
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MloK1 tetramer

EntireName: MloK1 tetramer
Components
  • Complex: MloK1 tetramer
    • Protein or peptide: Cyclic nucleotide-gated potassium channel mll3241
  • Ligand: POTASSIUM IONPotassium

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Supramolecule #1: MloK1 tetramer

SupramoleculeName: MloK1 tetramer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Cyclic nucleotide-modulated potassium channel in the presence of cAMP ligand, reconstituted into 2D lipid membrane crystals.
Source (natural)Organism: Mesorhizobium loti (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pASK90
Molecular weightTheoretical: 148 KDa

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Macromolecule #1: Cyclic nucleotide-gated potassium channel mll3241

MacromoleculeName: Cyclic nucleotide-gated potassium channel mll3241 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Mesorhizobium loti MAFF303099 (bacteria) / Organ: Membrane
Molecular weightTheoretical: 37.766297 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSVLPFLRIY APLNAVLAAP GLLAVAALTI PDMSGRSRLA LAALLAVIWG AYLLQLAATL LKRRAGVVRD RTPKIAIDVL AVLVPLAAF LLDGSPDWSL YCAVWLLKPL RDSTFFPVLG RVLANEARNL IGVTTLFGVV LFAVALAAYV IERDIQPEKF G SIPQAMWW ...String:
MSVLPFLRIY APLNAVLAAP GLLAVAALTI PDMSGRSRLA LAALLAVIWG AYLLQLAATL LKRRAGVVRD RTPKIAIDVL AVLVPLAAF LLDGSPDWSL YCAVWLLKPL RDSTFFPVLG RVLANEARNL IGVTTLFGVV LFAVALAAYV IERDIQPEKF G SIPQAMWW AVVTLSTTGY GDTIPQSFAG RVLAGAVMMS GIGIFGLWAG ILATGFYQEV RRGDFVRNWQ LVAAVPLFQK LG PAVLVEI VRALRARTVP AGAVICRIGE PGDRMFFVVE GSVSVATPNP VELGPGAFFG EMALISGEPR SATVSAATTV SLL SLHSAD FQMLCSSSPE IAEIFRKTAL ERRGAAASA

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Macromolecule #2: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 2 / 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
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

Concentration0.7 mg/mL
BufferpH: 7.6
Details: 20 mM KCl, 20 mM Tris-HCl pH 7.6, 1 mM BaCl2, 1 mM EDTA, 0.2 mM cAMP
GridModel: Quantifoil R3.5/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 3.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: 3.5 second-blotting.
Crystal formationLipid protein ratio: 0.8 / Lipid mixture: E.coli polar lipids / Instrument: dialysis buttons / Atmosphere: dialysis buffer / Temperature: 293.0 K / Time: 5.0 DAY
Details: DM solubilized MloK1 sample was mixed with E. coli polar lipid extract (Avanti Polar Lipids) at a lipid to protein ratio of 0.8 and dialyzed against detergent free buffer. 2D crystals of the ...Details: DM solubilized MloK1 sample was mixed with E. coli polar lipid extract (Avanti Polar Lipids) at a lipid to protein ratio of 0.8 and dialyzed against detergent free buffer. 2D crystals of the lipid embedded protein were obtained within 5 days.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.3 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 50000 / Camera length: 800 mm
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN / Tilt angle: 0., 55.
Detailspixel size 1.3 A/pix
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 30 / Number real images: 346 / Average exposure time: 16.0 sec. / Average electron dose: 45.0 e/Å2
Details: Each image was dose-fractionated in 40 frames (16 sec in total, 0.4-sec frames). The dose rate was set to ~5 counts/sec/physical-pixel (~2.8 e-/s/A2)leading to a total dose of ~45 e-/A2. Pixel size was 1.3A/pix.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Crystal parametersUnit cell - A: 135 Å / Unit cell - B: 135 Å / Unit cell - C: 200 Å / Unit cell - C sampling length: 135 Å / Unit cell - γ: 90 ° / Plane group: P 4 21 2
Crystallography statisticsNumber intensities measured: 6361 / Number structure factors: 6901357 / Fourier space coverage: 100 / R sym: 99 / R merge: 99 / Overall phase error: 99 / Overall phase residual: 99 / Phase error rejection criteria: 99 / High resolution: 4.5 Å
Details: Image processing was done with FOCUS (formerly 2dx), available at FOCUS-EM.org. Arheit et al., 2013a; Arheit et al., 2013b; Arheit et al., 2013c; Gipson et al., 2008; Gipson et al., 2007; ...Details: Image processing was done with FOCUS (formerly 2dx), available at FOCUS-EM.org. Arheit et al., 2013a; Arheit et al., 2013b; Arheit et al., 2013c; Gipson et al., 2008; Gipson et al., 2007; Gipson et al., 2011; Biyani et al., 2017. Rsym, Rmerge, and phase errors are not available.
Shell - Shell ID: 1 / Shell - High resolution: 4.5 Å / Shell - Low resolution: 6.0 Å / Shell - Number structure factors: 6901357 / Shell - Phase residual: 99 / Shell - Fourier space coverage: 100 / Shell - Multiplicity: 300
CTF correctionSoftware - Version: 2.1 / Software - details: MotionCor 2.1
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: OTHER
Merging software listSoftware - details: Arheit et al., 2013a; Arheit et al., 2013b; Arheit et al., 2013c; Gipson et al., 2008; Gipson et al., 2007; Gipson et al., 2011; Biyani et al., 2017
DetailsGatan Quantum-LS energy filter, with K2 Summit detector

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Atomic model buiding 1

Initial modelPDB ID:

4chv


Chain - Chain ID: A / Chain - Residue range: 1-355
DetailsThe initial model was obtained using Modeller (Sali and Blundell, 1993); in particular, the missing fragments were generated for the previously published PDB 4CHV model. This starting model was refined using the Rosetta for cryo-EM package (DiMaio et al., 2015). The symmetry of the channel was restrained during optimization runs (performed following the package tutorial (Wang and DiMaio,2015)). A model with a high fit score to the cryo-EM map and a low energy, as defined by the Rosetta force field, was selected from 100 Rosetta models generated and refined further. Several rounds of manual refinement with Coot (Emsley et al.,2010) and global optimization with Phenix (real_space_refine method (Afonine et al., 2013)) were carried out. Secondary structure constraints were imposed to stabilize the fold of helices and b-sheets during the global optimization.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: fit energy
Output model

PDB-6eo1:
The electron crystallography structure of the cAMP-bound potassium channel MloK1 (PCO-refined)

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