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- EMDB-4439: Full MloK1 consensus map from single particle analysis of 2D crystals -

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Basic information

Entry
Database: EMDB / ID: EMD-4439
TitleFull MloK1 consensus map from single particle analysis of 2D crystals
Map dataFull MloK1 consensus map from single particle analysis of 2D crystals comprising 2x2 unit cells, sharpened.
Sample
  • Complex: MloK1 potassium channel in lipidic bilayer of 2D crystals
Biological speciesMesorhizobium loti MAFF303099 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 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
DepositionNov 27, 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: 0.055
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4439.png

Downloads & links

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Map

FileDownload / File: emd_4439.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull MloK1 consensus map from single particle analysis of 2D crystals comprising 2x2 unit cells, sharpened.
Voxel sizeX=Y=Z: 1.3 Å
Density
Contour LevelBy AUTHOR: 0.055 / Movie #1: 0.055
Minimum - Maximum-0.15064608 - 0.22165443
Average (Standard dev.)-0.0004648512 (±0.012156556)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 416.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z416.000416.000416.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1510.222-0.000

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Supplemental data

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Half map: #1

Fileemd_4439_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_4439_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

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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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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 angle assignmentType: OTHER / Software - Name: FREALIGN (ver. 9.11)
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11)
Details: Refinement resolution limit set to 7.52 A in FREALIGN. Map was sharpened by deconvolution of the detector MTF at 300 kV and negative B-factor application.
Number images used: 231688
FSC plot (resolution estimation)

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