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- PDB-6eo1: The electron crystallography structure of the cAMP-bound potassiu... -

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Basic information

Entry
Database: PDB / ID: 6eo1
TitleThe electron crystallography structure of the cAMP-bound potassium channel MloK1 (PCO-refined)
ComponentsCyclic nucleotide-gated potassium channel mll3241
KeywordsMEMBRANE PROTEIN / MloK1 / MlotiK1 / potassium channel / CNBD / cytoplasmic domains / PCO refinement
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 / Ion channel / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily ...Cyclic nucleotide-regulated ion channel, N-terminal / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Ion channel / 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 MAFF303099 (bacteria)
MethodELECTRON CRYSTALLOGRAPHY / electron 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.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation315230_146929, 205320_144427 Switzerland
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, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 27, 2017Provider: repository / Type: Initial release
Revision 1.1Jan 10, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: Cyclic nucleotide-gated potassium channel mll3241
B: Cyclic nucleotide-gated potassium channel mll3241
C: Cyclic nucleotide-gated potassium channel mll3241
D: Cyclic nucleotide-gated potassium channel mll3241
hetero molecules


Theoretical massNumber of molelcules
Total (without water)151,1436
Polymers151,0654
Non-polymers782
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14990 Å2
ΔGint-107 kcal/mol
Surface area73090 Å2
MethodPISA
Unit cell
Length a, b, c (Å)252.450, 252.450, 209.000
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number1
Space group name H-MP1

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Components

#1: Protein
Cyclic nucleotide-gated potassium channel mll3241 / MlotiK1 channel


Mass: 37766.297 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mesorhizobium loti MAFF303099 (bacteria)
Gene: mll3241 / Organ: MembraneBiological membrane / Plasmid: pASK90 / Cell (production host): E. coli / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q98GN8
#2: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography
Crystal symmetry∠γ: 90 ° / C sampling length: 135 Å / A: 135 Å / B: 135 Å / C: 200 Å / Space group name H-M: P4212

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Sample preparation

ComponentName: MloK1 tetramer / Type: COMPLEX
Details: Cyclic nucleotide-modulated potassium channel in the presence of cAMP ligand, reconstituted into 2D lipid membrane crystals.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.148 MDa / Experimental value: NO
Source (natural)Organism: Mesorhizobium loti (bacteria) / Organ: Membrane
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pASK90
EM crystal formationInstrument: dialysis buttons / Atmosphere: dialysis buffer
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.
Lipid mixture: E.coli polar lipids / Lipid protein ratio: 0.8 / Temperature: 293 K / Time: 5 DAY
Buffer solutionpH: 7.6
Details: 20 mM KCl, 20 mM Tris-HCl pH 7.6, 1 mM BaCl2, 1 mM EDTA, 0.2 mM cAMP
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R3.5/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 K / Details: 3.5 second-blotting

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Data collection

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: pixel size 1.3 A/pix
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Nominal defocus max: 4300 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 16 sec. / Electron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 30 / Num. of real 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.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansMovie frames/image: 40
EM diffractionCamera length: 800 mm / Tilt angle list: 0, 55
EM diffraction shellResolution: 4.5→6 Å / Fourier space coverage: 100 % / Multiplicity: 300 / Num. of structure factors: 6901357 / Phase residual: 99 °
EM diffraction statsDetails: 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.
Fourier space coverage: 100 % / High resolution: 4.5 Å / Num. of intensities measured: 6361 / Num. of structure factors: 6901357 / Phase error: 99 ° / Phase residual: 99 ° / Phase error rejection criteria: 99 / Rmerge: 99 / Rsym: 99

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1FOCUS1image acquisition
32.1CTF correctionMotionCor 2.1
6Rosettamodel fitting
8PHENIXmodel refinement
Image processingDetails: Gatan Quantum-LS energy filter, with K2 Summit detector
Crystal symmetry∠γ: 90 ° / C sampling length: 135 Å / A: 135 Å / B: 135 Å / C: 200 Å / Space group name H-M: P4212
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionMethod: CRYSTALLOGRAPHY / Resolution: 4.5 Å / Resolution method: OTHER / Symmetry type: 2D CRYSTAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: fit energy
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 ...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.
Atomic model buildingPDB-ID: 4CHV

4chv


Pdb chain-ID: A / Pdb chain residue range: 1-355
RefinementResolution: 4.5→6 Å / Cross valid method: NONE /
Num. reflection% reflection
obs23102 100 %
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.01314812
ELECTRON CRYSTALLOGRAPHYf_angle_d0.93423760
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d10.0499316
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.0471784
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0072372

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