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- PDB-5tr1: Cryo-electron microscopy structure of a bovine CLC-K chloride cha... -

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Basic information

Entry
Database: PDB / ID: 5tr1
TitleCryo-electron microscopy structure of a bovine CLC-K chloride channel, alternate (class 2) conformation
Components
  • Chloride channel protein
  • Monoclonal antibody, Fab fragment, heavy chain
  • Monoclonal antibody, Fab fragment, light chain
KeywordsTRANSPORT PROTEIN / CLC / chloride channel / membrane / kidney
Function / homology
Function and homology information


Stimuli-sensing channels / voltage-gated chloride channel activity / chloride transport / membrane
Similarity search - Function
Chloride channel ClC-K / CBS-domain / CBS-domain / Chloride channel, voltage gated / Chloride channel, core / Voltage gated chloride channel / CBS domain superfamily / Immunoglobulins / Roll / Immunoglobulin-like ...Chloride channel ClC-K / CBS-domain / CBS-domain / Chloride channel, voltage gated / Chloride channel, core / Voltage gated chloride channel / CBS domain superfamily / Immunoglobulins / Roll / Immunoglobulin-like / Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
CHOLESTEROL HEMISUCCINATE / Uncharacterized protein
Similarity search - Component
Biological speciesBos taurus (cattle)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.95 Å
AuthorsPark, E. / MacKinnon, R.
Funding support United States, 2items
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 25, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 11, 2017Provider: repository / Type: Initial release
Revision 1.1Feb 8, 2017Group: Database references
Revision 1.2Sep 27, 2017Group: Author supporting evidence / Data collection / Experimental preparation
Category: em_image_scans / em_sample_support ...em_image_scans / em_sample_support / em_software / pdbx_audit_support
Item: _em_sample_support.grid_type / _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.4Nov 6, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB / _cell.length_a / _cell.length_b / _cell.length_c
Revision 1.5Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Chloride channel protein
H: Monoclonal antibody, Fab fragment, heavy chain
L: Monoclonal antibody, Fab fragment, light chain
B: Chloride channel protein
I: Monoclonal antibody, Fab fragment, heavy chain
M: Monoclonal antibody, Fab fragment, light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)196,9318
Polymers195,9576
Non-polymers9732
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12L
22M
13H
23I

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1111A48 - 683
2111B48 - 683
1121L1 - 107
2121M1 - 107
1131H1 - 111
2131I1 - 111

NCS ensembles :
ID
1
2
3

NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(-1, 0.0001, -3.5E-5), (-0.0001, -1, 1.6E-5), (-3.5E-5, 1.6E-5, 1)109.19422, 116.98939, 0.00211
3given(1), (1), (1)
4given(-1, 7.9E-5, 2.7E-5), (-7.9E-5, -1, -0.00011), (2.7E-5, -0.00011, 1)109.19074, 117.00269, 0.00605
5given(1), (1), (1)
6given(-1, -6.0E-6, 4.6E-5), (6.0E-6, -1, -5.4E-5), (4.6E-5, -5.4E-5, 1)109.19421, 116.98991, 0.00201

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Components

#1: Protein Chloride channel protein /


Mass: 73521.484 Da / Num. of mol.: 2 / Mutation: N373Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: LOC101909378 / Cell line (production host): HEK292S GnTI- / Production host: Homo sapiens (human) / References: UniProt: E1B792
#2: Antibody Monoclonal antibody, Fab fragment, heavy chain /


Mass: 12633.068 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Cell line: Hybridoma 25E7
#3: Antibody Monoclonal antibody, Fab fragment, light chain /


Mass: 11824.102 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)
#4: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C31H50O4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: A bovine CLC-K channel complexed with a monoclonal antibody fragment (Fab)
Type: COMPLEX
Details: Fab fragment generated by proteolytic (papain) cleavage of murine IgG antibody.
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bos taurus (cattle)
Source (recombinant)Organism: Homo sapiens (human) / Plasmid: pFastBac (BacMam)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaClSodium chloride1
220 mMTrisC4H11NO31
31 mMDL-dithiothreitolC4H10O2S21
40.04 %n-dodecyl-beta-D-maltosideC24H46O111
50.004 %cholestery hemisuccinateC3H50O41
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 295 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 0.3 sec. / Electron dose: 1.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0088 / Classification: refinement
EM software
IDNameVersionCategory
1RELION1.4particle selection
4CTFFIND4CTF correction
7Coot0.8.1model fitting
9REFMAC5.8model refinement
10RELION1.4initial Euler assignment
11RELION1.4final Euler assignment
12RELION1.4classification
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67475
Details: the indicated resolution was estimated on the primary map using the provided mask including Fab.
Symmetry type: POINT
Refinement stepCycle: 1 / Total: 12678
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01913024
ELECTRON MICROSCOPYr_bond_other_d0.0020.0212370
ELECTRON MICROSCOPYr_angle_refined_deg1.411.95517760
ELECTRON MICROSCOPYr_angle_other_deg0.957328366
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.75551634
ELECTRON MICROSCOPYr_dihedral_angle_2_deg28.13122.797472
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.167151986
ELECTRON MICROSCOPYr_dihedral_angle_4_deg10.5561556
ELECTRON MICROSCOPYr_chiral_restr0.0990.22058
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.02114494
ELECTRON MICROSCOPYr_gen_planes_other0.0010.023068
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it9.33732.0846578
ELECTRON MICROSCOPYr_mcbond_other9.31432.0846577
ELECTRON MICROSCOPYr_mcangle_it15.12548.1218198
ELECTRON MICROSCOPYr_mcangle_other15.12548.1228199
ELECTRON MICROSCOPYr_scbond_it10.31433.3666446
ELECTRON MICROSCOPYr_scbond_other10.31333.3676447
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other17.70149.5859563
ELECTRON MICROSCOPYr_long_range_B_refined23.54424028
ELECTRON MICROSCOPYr_long_range_B_other23.54424028
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Dom-ID: 1 / Refine-ID: ELECTRON MICROSCOPY / Type: tight thermal / Weight position: 0.5

Ens-IDAuth asym-IDNumberRms dev position (Å)
1A89851.18
2L15970.68
3H16572.18
LS refinement shellResolution: 3.9→4.001 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.581 4545 -
Rfree-0 -
obs--100 %

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