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- EMDB-8454: Cryo-electron microscopy structure of a bovine CLC-K chloride cha... -

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Basic information

Entry
Database: EMDB / ID: EMD-8454
TitleCryo-electron microscopy structure of a bovine CLC-K chloride channel, alternate (class 2) conformation
Map data
SampleA bovine CLC-K channel complexed with a monoclonal antibody fragment (Fab)
  • Chloride channel protein
  • (Monoclonal antibody, Fab fragment, ...) x 2
  • ligand
Function / homologyCBS domain / Chloride channel, voltage gated / Chloride channel ClC-K / Chloride channel, core / voltage-gated chloride channel activity / chloride transport / integral component of plasma membrane / Chloride channel protein
Function and homology information
Biological speciesBos taurus (cattle) / house mouse (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.95 Å
AuthorsPark E / MacKinnon R
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Jane Coffin Childs Memorial Fund61-1513 United States
CitationJournal: Nature / Year: 2017
Title: Structure of a CLC chloride ion channel by cryo-electron microscopy.
Authors: Eunyong Park / Ernest B Campbell / Roderick MacKinnon /
Abstract: CLC proteins transport chloride (Cl) ions across cellular membranes to regulate muscle excitability, electrolyte movement across epithelia, and acidification of intracellular organelles. Some CLC ...CLC proteins transport chloride (Cl) ions across cellular membranes to regulate muscle excitability, electrolyte movement across epithelia, and acidification of intracellular organelles. Some CLC proteins are channels that conduct Cl ions passively, whereas others are secondary active transporters that exchange two Cl ions for one H. The structural basis underlying these distinctive transport mechanisms is puzzling because CLC channels and transporters are expected to share the same architecture on the basis of sequence homology. Here we determined the structure of a bovine CLC channel (CLC-K) using cryo-electron microscopy. A conserved loop in the Cl transport pathway shows a structure markedly different from that of CLC transporters. Consequently, the cytosolic constriction for Cl passage is widened in CLC-K such that the kinetic barrier previously postulated for Cl/H transporter function would be reduced. Thus, reduction of a kinetic barrier in CLC channels enables fast flow of Cl down its electrochemical gradient.
Validation ReportPDB-ID: 5tr1

SummaryFull reportAbout validation report
History
DepositionOct 26, 2016-
Header (metadata) releaseDec 28, 2016-
Map releaseJan 11, 2017-
UpdateNov 20, 2019-
Current statusNov 20, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0176
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0176
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5tr1
  • Surface level: 0.0176
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8454.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 240 pix.
= 312. Å
1.3 Å/pix.
x 240 pix.
= 312. Å
1.3 Å/pix.
x 240 pix.
= 312. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3 Å
Density
Contour LevelBy AUTHOR: 0.0176 / Movie #1: 0.0176
Minimum - Maximum-0.024342816 - 0.056552105
Average (Standard dev.)0.00012446755 (±0.0026305348)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 312.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z312.000312.000312.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0240.0570.000

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Supplemental data

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Segmentation: #1

Fileemd_8454_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: B-factor-sharpend (-120 A^2) and lowpass-filtered (4.1 Ang)

Fileemd_8454_additional_1.map
AnnotationB-factor-sharpend (-120 A^2) and lowpass-filtered (4.1 Ang)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: B-factor-sharpend (-120 A^2) and lowpass-filtered (3.9 Ang)

Fileemd_8454_additional_2.map
AnnotationB-factor-sharpend (-120 A^2) and lowpass-filtered (3.9 Ang)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire A bovine CLC-K channel complexed with a monoclonal antibody fragm...

EntireName: A bovine CLC-K channel complexed with a monoclonal antibody fragment (Fab)
Details: Fab fragment generated by proteolytic (papain) cleavage of murine IgG antibody.
Number of components: 5

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Component #1: protein, A bovine CLC-K channel complexed with a monoclonal antib...

ProteinName: A bovine CLC-K channel complexed with a monoclonal antibody fragment (Fab)
Details: Fab fragment generated by proteolytic (papain) cleavage of murine IgG antibody.
Recombinant expression: No
SourceSpecies: Bos taurus (cattle)
Source (engineered)Expression System: Homo sapiens (human) / Vector: pFastBac (BacMam)

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Component #2: protein, Chloride channel protein

ProteinName: Chloride channel protein / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 73.521484 kDa
SourceSpecies: Bos taurus (cattle)
Source (engineered)Expression System: Homo sapiens (human)

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Component #3: protein, Monoclonal antibody, Fab fragment, heavy chain

ProteinName: Monoclonal antibody, Fab fragment, heavy chain / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 12.633068 kDa
SourceSpecies: house mouse (house mouse)

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Component #4: protein, Monoclonal antibody, Fab fragment, light chain

ProteinName: Monoclonal antibody, Fab fragment, light chain / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 11.824102 kDa
SourceSpecies: house mouse (house mouse)

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Component #5: ligand, CHOLESTEROL HEMISUCCINATE

LigandName: CHOLESTEROL HEMISUCCINATE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.486726 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 3 mg/mL / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 295 K / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.8 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 67475
3D reconstructionSoftware: RELION / Resolution: 3.95 Å / Resolution method: FSC 0.143 CUT-OFF
Details: the indicated resolution was estimated on the primary map using the provided mask including Fab.
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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