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- PDB-6i9d: MloK1 consensus structure from single particle analysis of 2D crystals -

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Basic information

Entry
Database: PDB / ID: 6i9d
TitleMloK1 consensus structure from single particle analysis of 2D crystals
ComponentsCyclic nucleotide-gated potassium channel mll3241
KeywordsMEMBRANE PROTEIN / Voltage-gated potassium channel / Cyclic nucleotide-binding domain / ion channel / ion transport
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 japonicum
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsRighetto, R. / Biyani, N. / Kowal, J. / Chami, M. / Stahlberg, H.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation205320_166164 Switzerland
CitationJournal: Nat Commun / Year: 2019
Title: Retrieving high-resolution information from disordered 2D crystals by single-particle cryo-EM.
Authors: Ricardo D Righetto / Nikhil Biyani / Julia Kowal / Mohamed Chami / Henning Stahlberg /
Abstract: Electron crystallography can reveal the structure of membrane proteins within 2D crystals under close-to-native conditions. High-resolution structural information can only be reached if crystals are ...Electron crystallography can reveal the structure of membrane proteins within 2D crystals under close-to-native conditions. High-resolution structural information can only be reached if crystals are perfectly flat and highly ordered. In practice, such crystals are difficult to obtain. Available image unbending algorithms correct for disorder, but only perform well on images of non-tilted, flat crystals, while out-of-plane distortions are not addressed. Here, we present an approach that employs single-particle refinement procedures to locally unbend crystals in 3D. With this method, density maps of the MloK1 potassium channel with a resolution of 4 Å were obtained from images of 2D crystals that do not diffract beyond 10 Å. Furthermore, 3D classification allowed multiple structures to be resolved, revealing a series of MloK1 conformations within a single 2D crystal. This conformational heterogeneity explains the poor diffraction observed and is related to channel function. The approach is implemented in the FOCUS package.
History
DepositionNov 23, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 24, 2019Provider: repository / Type: Initial release

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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
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18370 Å2
ΔGint-151 kcal/mol
Surface area68110 Å2
MethodPISA

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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 japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099) (bacteria)
Strain: LMG 29417 / CECT 9101 / MAFF 303099 / Gene: mll3241 / 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 MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: MloK1 potassium channel in lipidic bilayer of 2D crystals
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.16 MDa / Experimental value: YES
Source (natural)Organism: Mesorhizobium loti MAFF303099 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
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
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Nominal defocus max: 4300 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm
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 real images: 270
Details: The dataset of 346 movies recorded and processed for Kowal et al. (2018) 2 was employed. As reported, the data were collected on an FEI Titan Krios TEM equipped with a Gatan K2 DED. Total ...Details: The dataset of 346 movies recorded and processed for Kowal et al. (2018) 2 was employed. As reported, the data were collected on an FEI Titan Krios TEM equipped with a Gatan K2 DED. Total dose: 40 e-/A^2 distributed over 40 movie frames. Pixel size: 1.3 A on the sample level (counting mode). Nominal tilt range: -55 to +55 degrees.

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Processing

EM software
IDNameVersionCategory
4CTFFINDCTF correction
5CTFTILTCTF correction
8iMODFIT1.44model fitting
10FOCUSinitial Euler assignment
11FREALIGN9.11final Euler assignment
13FREALIGN9.113D reconstruction
14PHENIX1.13model refinement
CTF correctionDetails: The defocus at the center of each particle box was estimated based on the tilt geometry.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 231688
Details: The particle positions were given by the cross-correlation profile generated by the classical unbending algorithm.
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 231688 / Algorithm: FOURIER SPACE
Details: Angular assignments and x,y shifts were refined using a custom auto-refinement script written for FREALIGN v9.11. Maximum resolution used for refinement was 7.52 A.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
16EO1

6eo1

A11-6
23BEH

3beh

A17-219
33CL1

3cl1

A1220-349
46EO1

6eo1

A1350-355

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