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

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Basic information

Database: EMDB / ID: 3907
TitleThe electron crystallography structure of the cAMP-bound potassium channel MloK1 (PCO-refined)
SampleMloK1 tetramer
SourceMesorhizobium loti / bacteria / メソリゾビウム・ロティ
Map dataPCO (Projective ConstraintOptimization) - refined 3D volume of MloK1 with cAMP
Methodelectron crystallography, at 4.5 Å resolution
AuthorsKowal J / Biyani N
CitationStructure, 2018, 26, 20-27.e3

Structure, 2018, 26, 20-27.e3 Yorodumi Papers
High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer.
Julia Kowal / Nikhil Biyani / Mohamed Chami / Sebastian Scherer / Andrzej J Rzepiela / Paul Baumgartner / Vikrant Upadhyay / Crina M Nimigean / Henning Stahlberg

Validation ReportPDB-ID: 6eo1

SummaryFull reportAbout validation report
DateDeposition: Oct 8, 2017 / Header (metadata) release: Dec 20, 2017 / Map release: Dec 27, 2017 / Last update: Jan 10, 2018

Structure visualization

  • 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
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6eo1
  • Surface level: 600
  • Imaged by UCSF CHIMERA
  • Download
3D viewer

View / / Stereo:
Slabnear <=> far

fix: /
Orientation Rotation
Misc. /
Supplemental images

Downloads & links


Fileemd_3907.map.gz (map file in CCP4 format, 10086 KB)
Projections & slices

Image control

AxesZ (Sec.)Y (Row.)X (Col.)
201 pix
1.05 Å/pix.
= 210.045 Å
112 pix
0.96 Å/pix.
= 106.96 Å
112 pix
0.96 Å/pix.
= 106.96 Å



Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

(generated in cubic-lattice coordinate)

Voxel sizeX: 0.955 Å / Y: 0.955 Å / Z: 1.045 Å
Contour Level:600 (by author), 600 (movie #1):
Minimum - Maximum-499.99753 - 2773.677
Average (Standard dev.)9.9917555 (257.0787)


Space Group Number1
Map Geometry
Axis orderXYZ
CellA: 106.96 Å / B: 106.96 Å / C: 210.045 Å
α=β=γ: 90 deg.

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
start NX/NY/NZ
MAP C/R/S123
start NC/NR/NS550
D min/max/mean-499.9982773.6779.992

Supplemental data

Sample components

Entire MloK1 tetramer

EntireName: MloK1 tetramer
Details: Cyclic nucleotide-modulated potassium channel in the presence of cAMP ligand, reconstituted into 2D lipid membrane crystals.
Number of components: 3
MassTheoretical: 148 kDa

Component #1: protein, MloK1 tetramer

ProteinName: MloK1 tetramer
Details: Cyclic nucleotide-modulated potassium channel in the presence of cAMP ligand, reconstituted into 2D lipid membrane crystals.
Recombinant expression: No
MassTheoretical: 148 kDa
SourceSpecies: Mesorhizobium loti / bacteria / メソリゾビウム・ロティ
Source (engineered)Expression System: Escherichia coli bl21(de3) / bacteria / image: Escherichia coli
Vector: pASK90

Component #2: protein, Cyclic nucleotide-gated potassium channel mll3241

ProteinName: Cyclic nucleotide-gated potassium channel mll3241 / Recombinant expression: No
MassTheoretical: 37.766297 kDa
Source (engineered)Expression System: Mesorhizobium loti maff303099 / bacteria / Vector: pASK90

Component #3: ligand, POTASSIUM ION

LigandName: POTASSIUM ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 3.909805 MDa

Experimental details

Sample preparation

Specimen state2D array
Crystal parametersPlane group: P 4 21 2 / A: 135 Å / B: 135 Å / C: 200 Å / Gamma: 90 deg.
Crystal grow detailsDM 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.
Sample solutionSpecimen conc.: 0.7 mg/ml
Buffer solution: 20 mM KCl, 20 mM Tris-HCl pH 7.6, 1 mM BaCl2, 1 mM EDTA, 0.2 mM cAMP
pH: 7.6
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 293 K / Humidity: 90 % / Details: 3.5 second-blotting

Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Details: pixel size 1.3 A/pix
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 45 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 50000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 750 - 4300 nm / Energy filter: GIF Quantum LS / Energy window: 0-20 eV
CameraDetector: GATAN K2 (4k x 4k)

Image acquisition

Image acquisitionNumber of digital images: 346
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.

Image processing

ProcessingMethod: electron crystallography
Details: Gatan Quantum-LS energy filter, with K2 Summit detector
3D reconstructionResolution: 4.5 Å / Resolution method: OTHER

Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: fit energy / Refinement space: REAL
Details: The 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.
Input PDB model: 4CHV
Chain ID: 4CHV_A
Output model

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