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- EMDB-4517: MloK1 map from single particle analysis of 2D crystals, class 6 (... -

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Basic information

Entry
Database: EMDB / ID: EMD-4517
TitleMloK1 map from single particle analysis of 2D crystals, class 6 (intermediate compact conformation)
Map data
Sample
  • Complex: MloK1 potassium channel in lipidic bilayer of 2D crystals
    • Protein or peptide: Cyclic nucleotide-gated potassium channel mll3241
  • Ligand: POTASSIUM IONPotassium
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 / 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 ...Cyclic nucleotide-regulated ion channel, N-terminal / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsRighetto R / Biyani N / Kowal J / Chami M / Stahlberg H
Funding support Switzerland, 1 items
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
DepositionDec 31, 2018-
Header (metadata) releaseApr 24, 2019-
Map releaseApr 24, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6qd2
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6qd2
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4517.png

Downloads & links

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Map

FileDownload / File: emd_4517.map.gz / Format: CCP4 / Size: 4.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.3 Å
Density
Contour LevelBy AUTHOR: 2 / Movie #1: 2
Minimum - Maximum-5.2331505 - 7.196864
Average (Standard dev.)0.00000000004 (±1)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions104104104
Spacing104104104
CellA=B=C: 135.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z104104104
origin x/y/z0.0000.0000.000
length x/y/z135.200135.200135.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS104104104
D min/max/mean-5.2337.1970.000

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Supplemental data

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Mask #1

Fileemd_4517_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_4517_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_4517_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MloK1 potassium channel in lipidic bilayer of 2D crystals

EntireName: MloK1 potassium channel in lipidic bilayer of 2D crystals
Components
  • Complex: MloK1 potassium channel in lipidic bilayer of 2D crystals
    • Protein or peptide: Cyclic nucleotide-gated potassium channel mll3241
  • Ligand: POTASSIUM IONPotassium

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Supramolecule #1: MloK1 potassium channel in lipidic bilayer of 2D crystals

SupramoleculeName: MloK1 potassium channel in lipidic bilayer of 2D crystals
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mesorhizobium loti MAFF303099 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightExperimental: 160 KDa

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Macromolecule #1: Cyclic nucleotide-gated potassium channel mll3241

MacromoleculeName: Cyclic nucleotide-gated potassium channel mll3241 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Mesorhizobium loti MAFF303099 (bacteria)
Molecular weightTheoretical: 37.766297 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSVLPFLRIY APLNAVLAAP GLLAVAALTI PDMSGRSRLA LAALLAVIWG AYLLQLAATL LKRRAGVVRD RTPKIAIDVL AVLVPLAAF LLDGSPDWSL YCAVWLLKPL RDSTFFPVLG RVLANEARNL IGVTTLFGVV LFAVALAAYV IERDIQPEKF G SIPQAMWW ...String:
MSVLPFLRIY APLNAVLAAP GLLAVAALTI PDMSGRSRLA LAALLAVIWG AYLLQLAATL LKRRAGVVRD RTPKIAIDVL AVLVPLAAF LLDGSPDWSL YCAVWLLKPL RDSTFFPVLG RVLANEARNL IGVTTLFGVV LFAVALAAYV IERDIQPEKF G SIPQAMWW AVVTLSTTGY GDTIPQSFAG RVLAGAVMMS GIGIFGLWAG ILATGFYQEV RRGDFVRNWQ LVAAVPLFQK LG PAVLVEI VRALRARTVP AGAVICRIGE PGDRMFFVVE GSVSVATPNP VELGPGAFFG EMALISGEPR SATVSAATTV SLL SLHSAD FQMLCSSSPE IAEIFRKTAL ERRGAAASA

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Macromolecule #2: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state2D array

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Sample preparation

BufferpH: 7.6
Details: 20 mM KCl, 20 mM Tris-HCl pH 7.6, 1 mM BaCl2, 1 mM EDTA, 0.2 mM cAMP
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.3 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 270 / Average exposure time: 16.0 sec. / Average electron dose: 45.0 e/Å2
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.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 231688
Details: The particle positions were given by the cross-correlation profile generated by the classical unbending algorithm.
CTF correctionSoftware: (Name: CTFFIND, CTFTILT)
Details: The defocus at the center of each particle box was estimated based on the tilt geometry.
Startup modelType of model: OTHER
Details: An initial model was generated using the tilt geometry determined by the classical 2D electron crystallography approach.
Initial angle assignmentType: OTHER / Software - Name: FOCUS
Details: An initial model was generated using the tilt geometry determined by the classical 2D electron crystallography approach.
Final 3D classificationNumber classes: 8 / Avg.num./class: 28961 / Software - Name: FREALIGN (ver. 9.11)
Final angle assignmentType: OTHER / Software - Name: FREALIGN (ver. 9.11)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Number images used: 22010
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6i9d

DetailsThe consensus model was flexibily fit in to the EM map of this class using Normal Mode Analysis with iMODFIT, and then real-space refined using PHENIX.
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6qd2:
MloK1 model from single particle analysis of 2D crystals, class 6 (intermediate compact conformation)

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