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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: 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
SampleMloK1 tetramer
  • Cyclic nucleotide-gated potassium channel mll3241
  • ligand
Function / homologyCyclic nucleotide-binding-like / Cyclic nucleotide-binding domain signature 1. / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain / Potassium channel domain / RmlC-like jelly roll fold / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-regulated ion channel, N-terminal / Voltage-gated potassium channel ...Cyclic nucleotide-binding-like / Cyclic nucleotide-binding domain signature 1. / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain / Potassium channel domain / RmlC-like jelly roll fold / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-regulated ion channel, N-terminal / Voltage-gated potassium channel / Cyclic nucleotide-binding domain / Ion channel / voltage-gated potassium channel complex / voltage-gated potassium channel activity / cAMP binding / identical protein binding / Cyclic nucleotide-gated potassium channel mll3241
Function and homology information
SourceMesorhizobium loti (bacteria)
Methodelectron crystallography / cryo EM / 4.5 Å resolution
AuthorsKowal J / Biyani N
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.
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

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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
  • Download
  • 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

Downloads & links

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Map

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

Image control

Size
Brightness
Contrast
Others
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

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

(generated in cubic-lattice coordinate)

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

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions112112201
Origin550
Limit116116200
Spacing112112201
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
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
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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Sample components

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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

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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)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pASK90

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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

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Component #3: ligand, POTASSIUM ION

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

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Experimental details

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Sample preparation

SpecimenSpecimen state: 2D array / Method: cryo EM
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

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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
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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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.

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Image processing

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

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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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