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- EMDB-0474: cryoEM map of NKCC1 -

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Basic information

Entry
Database: EMDB / ID: EMD-0474
TitlecryoEM map of NKCC1
Map datacryoEM map of NKCC1
Sample
  • Organelle or cellular component: protein sample
Biological speciesSpodoptera frugiperda (fall armyworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsBenjamin O
CitationJournal: Nature / Year: 2019
Title: Structure and mechanism of the cation-chloride cotransporter NKCC1.
Authors: Thomas A Chew / Benjamin J Orlando / Jinru Zhang / Naomi R Latorraca / Amy Wang / Scott A Hollingsworth / Dong-Hua Chen / Ron O Dror / Maofu Liao / Liang Feng /
Abstract: Cation-chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium and/or sodium across the membrane. They have critical roles in regulating cell volume, controlling ...Cation-chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium and/or sodium across the membrane. They have critical roles in regulating cell volume, controlling ion absorption and secretion across epithelia, and maintaining intracellular chloride homeostasis. These transporters are primary targets for some of the most commonly prescribed drugs. Here we determined the cryo-electron microscopy structure of the Na-K-Cl cotransporter NKCC1, an extensively studied member of the CCC family, from Danio rerio. The structure defines the architecture of this protein family and reveals how cytosolic and transmembrane domains are strategically positioned for communication. Structural analyses, functional characterizations and computational studies reveal the ion-translocation pathway, ion-binding sites and key residues for transport activity. These results provide insights into ion selectivity, coupling and translocation, and establish a framework for understanding the physiological functions of CCCs and interpreting disease-related mutations.
History
DepositionJan 17, 2019-
Header (metadata) releaseMar 27, 2019-
Map releaseSep 18, 2019-
UpdateSep 18, 2019-
Current statusSep 18, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0456
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0456
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0474.png

Downloads & links

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Map

FileDownload / File: emd_0474.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoEM map of NKCC1
Voxel sizeX=Y=Z: 1.055 Å
Density
Contour LevelBy AUTHOR: 0.0456 / Movie #1: 0.0456
Minimum - Maximum-0.08095613 - 0.13459511
Average (Standard dev.)-0.0005799969 (±0.0054328735)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 202.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0551.0551.055
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z202.560202.560202.560
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-0.0810.135-0.001

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Supplemental data

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Sample components

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Entire : protein sample

EntireName: protein sample
Components
  • Organelle or cellular component: protein sample

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Supramolecule #1: protein sample

SupramoleculeName: protein sample / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Spodoptera frugiperda (fall armyworm)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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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

Concentration7.9 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 96 % / Chamber temperature: 293 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 53.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 227231
FSC plot (resolution estimation)

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