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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)
DescriptorCyclic nucleotide-gated potassium channel mll3241
KeywordsMEMBRANE PROTEIN / MloK1 / MlotiK1 / potassium channel / CNBD / cytoplasmic domains / PCO refinement
Specimen sourceMesorhizobium loti maff303099 / bacteria
MethodElectron crystallography (4.5 Å resolution / 2d array / Crystallography)
AuthorsKowal, J. / Biyani, N. / Chami, M. / Scherer, S. / Rzepiela, A. / Baumgartner, P. / Upadhyay, V. / Nimigean, C. / Stahlberg, H.
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 Report
SummaryFull reportAbout validation report
DateDeposition: Oct 8, 2017 / Release: Dec 27, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 27, 2017Structure modelrepositoryInitial release
1.1Jan 10, 2018Structure modelDatabase referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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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
Polyers151,0654
Non-polymers782
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)14990
ΔGint (kcal/M)-107
Surface area (Å2)73090
MethodPISA
Unit cell
γ
α
β
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-MP 1

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Components

#1: Polypeptide(L)
Cyclic nucleotide-gated potassium channel mll3241 / MlotiK1 channel


Mass: 37766.297 Da / Num. of mol.: 4
Source: (gene. exp.) Mesorhizobium loti maff303099 / bacteria
References: UniProt: Q98GN8
#2: ChemicalChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Formula: K

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 2D ARRAY / Reconstruction method: CRYSTALLOGRAPHY
Crystal symmetryAngle gamma: 90 deg. / C sampling length: 135 Å / Length a: 135 Å / Length b: 135 Å / Length c: 200 Å / Space group name H-M: P 4 21 2

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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 deg. / Units: MEGADALTONS / Experimental value: NO
Source (natural)Organ: Membrane / Organism: Mesorhizobium loti
Source (recombinant)Organism: Escherichia coli BL21(DE3) / 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 lipid embedded protein were obtained within 5 days.
Lipid mixture: E.coli polar lipids / Lipid protein ratio: 0.8 / Temperature: 293 kelvins / Time: 5 sec. / Time unit: DAY
Buffer solutionDetails: 20 mM KCl, 20 mM Tris-HCl pH 7.6, 1 mM BaCl2, 1 mM EDTA, 0.2 mM cAMP
pH: 7.6
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 / Grid type: Quantifoil R3.5/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 kelvins / Details: 3.5 second-blotting

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Data collection

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: 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 FIELD / Nominal magnification: 50000 / Nominal defocus max: 4300 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 100 mm / 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
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.
Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 30 / Number of real images: 346
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 shellFourier space coverage: 100 / High resolution: 4.5 Å / Low resolution: 6 Å / Multiplicity: 300 / Number of structure factors: 6901357 / Phase residual: 99 deg.
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; 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 / Number of intensities measured: 6361 / Number of structure factors: 6901357 / Overall phase error: 99 deg. / Overall phase residual: 99 deg. / Phase error rejection criteria: 99 / R merge: 99 / R sym: 99

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategoryImaging IDDetailsImage processing IDFitting ID
1FOCUS1.0IMAGE ACQUISITION1
32.1CTF CORRECTIONMotionCor 2.11
6RosettaMODEL FITTING1
8PHENIXMODEL REFINEMENT1
Image processingDetails: Gatan Quantum-LS energy filter, with K2 Summit detector
Crystal symmetryAngle gamma: 90 deg. / C sampling length: 135 Å / Length a: 135 Å / Length b: 135 Å / Length c: 200 Å / Space group name H-M: P 4 21 2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionMethod: CRYSTALLOGRAPHY / Resolution: 4.5 Å / Resolution method: OTHER / Symmetry type: 2D CRYSTAL
Atomic model buildingDetails: 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.
Ref protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: fit energy
Atomic model buildingPDB-ID: 4CHV
Pdb chain ID: A / Pdb chain residue range: 1-355
RefineCross valid method: NONE
Least-squares processHighest resolution: 4.5 Å / Lowest resolution: 6 Å / Number reflection obs: 23102 / Percent reflection obs: 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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