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- EMDB-8435: Cryo-electron microscopy structure of a bovine CLC-K chloride cha... -

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Basic information

Entry
Database: EMDB / ID: EMD-8435
TitleCryo-electron microscopy structure of a bovine CLC-K chloride channel, main (class 1) conformation
Map dataStructure of a bovine CLC-K chloride channel. Combined, unfiltered map of Class 1
Sample
  • Complex: A bovine CLC-K channel complexed with a monoclonal antibody fragment (Fab)
    • Protein or peptide: Chloride channel protein
    • Protein or peptide: Monoclonal antibody, Fab fragment, light chain
    • Protein or peptide: Monoclonal antibody, Fab fragment, heavy chain
KeywordsCLC / chloride channel / membrane / kidney / TRANSPORT PROTEIN
Function / homology
Function and homology information


Stimuli-sensing channels / voltage-gated chloride channel activity / chloride transport / chloride channel complex / plasma membrane
Similarity search - Function
Chloride channel ClC-K / : / Chloride channel, voltage gated / Chloride channel, core / Voltage gated chloride channel / CBS domain superfamily
Similarity search - Domain/homology
Uncharacterized protein
Similarity search - Component
Biological speciesBos taurus (domestic cattle) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.76 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.
History
DepositionOct 26, 2016-
Header (metadata) releaseDec 28, 2016-
Map releaseJan 4, 2017-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.019
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5tqq
  • Surface level: 0.019
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8435.png

Downloads & links

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Map

FileDownload / File: emd_8435.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of a bovine CLC-K chloride channel. Combined, unfiltered map of Class 1
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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.019 / Movie #1: 0.019
Minimum - Maximum-0.022385363 - 0.065809295
Average (Standard dev.)0.000106467705 (±0.0027214182)
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-0.0220.0660.000

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Supplemental data

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Mask #1

Fileemd_8435_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: B-factor-sharpend (-100 A^2) and lowpass-filtered (3.7 Ang)

Fileemd_8435_additional_1.map
AnnotationB-factor-sharpend (-100 A^2) and lowpass-filtered (3.7 Ang)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: emd 8435 additional 2.map

Fileemd_8435_additional_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 : A bovine CLC-K channel complexed with a monoclonal antibody fragm...

EntireName: A bovine CLC-K channel complexed with a monoclonal antibody fragment (Fab)
Components
  • Complex: A bovine CLC-K channel complexed with a monoclonal antibody fragment (Fab)
    • Protein or peptide: Chloride channel protein
    • Protein or peptide: Monoclonal antibody, Fab fragment, light chain
    • Protein or peptide: Monoclonal antibody, Fab fragment, heavy chain

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Supramolecule #1: A bovine CLC-K channel complexed with a monoclonal antibody fragm...

SupramoleculeName: A bovine CLC-K channel complexed with a monoclonal antibody fragment (Fab)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Fab fragment generated by proteolytic (papain) cleavage of murine IgG antibody.
Source (natural)Organism: Bos taurus (domestic cattle)

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Macromolecule #1: Chloride channel protein

MacromoleculeName: Chloride channel protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 73.521484 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MRVRRGIRGG LDWLKRKLFC VGEDWYFLTV LGVLMALISF TMSFTVGRVV RAHKWLYREI GDSHLLRYLS WTVYPVALVS FSSGFSQSI TPFSGGSGIP ELKTILSGVV LEDYLDIKNF GAKAVGLTCT LASGSTIFLG KVGPFVHLSV MIAAYLGRVR A KATGESEN ...String:
MRVRRGIRGG LDWLKRKLFC VGEDWYFLTV LGVLMALISF TMSFTVGRVV RAHKWLYREI GDSHLLRYLS WTVYPVALVS FSSGFSQSI TPFSGGSGIP ELKTILSGVV LEDYLDIKNF GAKAVGLTCT LASGSTIFLG KVGPFVHLSV MIAAYLGRVR A KATGESEN KSKRNEMLVA GAAVGVATVF AAPFSGVLFC IEVVSSHFSV WDYWRGFFAA TCGAFMFRLL AVFNSEQETI TS LYKTSFR VEVPFDLPEI FFFVALGAIC GVASCAYLFC QRKFLGFVKT NPVLSKLMAT SKPLYSALAA LVLASVTYPP GAG RFMASR LSMREYLDSL LDHNSWALLT RQASPPWPVE PDPQNLWFEW YHPQFTIFGT LAFFLVMKFW MLILATTIPM PAGY FMPIF IFGAAIGRLL GEALSVAFPE GIVAGGVTNP IMPGGYALAG AAAFSGAVTH SISTALLAFE LTGQIVHALP VLMAV LAAN AIAQSCQPSF YDGTIIVKKL PYLPWIRGRK ISSHRVTVEH FMNRAITTLA KDTPQEEVVK VVTSTDMAEY PLVAST ESQ TLVGTMRRAQ LVQALQAEPP SWAPGQQRCL QDILAEGCPV EPVTLKLSPE TSLHQAHNLF ELLNLQSLFV TSQGRAV GF VSWVELEKAI SKLTNPPAPK SNSLEVLFQ

UniProtKB: Uncharacterized protein

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Macromolecule #2: Monoclonal antibody, Fab fragment, light chain

MacromoleculeName: Monoclonal antibody, Fab fragment, light chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 11.824102 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString:
DIVMTQSPKF MSTSVGDRVS VTCKASQNVG TNVAWYQQKP GQSPKTLIYW ASYRYSGVPD RFTGSGSGTD FTLAISNVQS EDLAEYFCQ QYNSYPLTFG SGTKLELK

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Macromolecule #3: Monoclonal antibody, Fab fragment, heavy chain

MacromoleculeName: Monoclonal antibody, Fab fragment, heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 12.633068 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString:
DVQLQESGPG LVKPSQSLSL TCTVTGDSVT SDYAWSWIRQ FPGKKLEWMG YITYSGNTIY NPSLKSRISI TRDTSKNQFF LQLKSVIIE DTATYYCSRG VDYWGQGTSV TVSS

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
100.0 mMNaClsodium chloride
20.0 mMC4H11NO3Tris
1.0 mMC4H10O2S2DL-dithiothreitol
0.04 %C24H46O11n-dodecyl-beta-D-maltoside
0.004 %C31H50O4cholesteryl hemisuccinate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average exposure time: 0.3 sec. / Average electron dose: 1.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Details: generated from EMAN2 and 2D averages of particle images
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4)
Details: the indicated resolution was estimated on the primary map using the provided mask including Fab.
Number images used: 82167
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.4)
Final 3D classificationSoftware - Name: RELION (ver. 1.4)
FSC plot (resolution estimation)

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