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- PDB-6wcb: Human open state TMEM175 in CsCl -

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Basic information

Entry
Database: PDB / ID: 6wcb
TitleHuman open state TMEM175 in CsCl
ComponentsEndosomal/lysosomal potassium channel TMEM175
KeywordsTRANSPORT PROTEIN / Ion Channel / Lysosome / Potassium Channel
Function / homology
Function and homology information


lysosomal lumen pH elevation / phagosome-lysosome fusion / regulation of lysosomal lumen pH / potassium ion leak channel activity / proton channel activity / arachidonate binding / potassium channel activity / potassium ion transmembrane transport / proton transmembrane transport / neuron cellular homeostasis ...lysosomal lumen pH elevation / phagosome-lysosome fusion / regulation of lysosomal lumen pH / potassium ion leak channel activity / proton channel activity / arachidonate binding / potassium channel activity / potassium ion transmembrane transport / proton transmembrane transport / neuron cellular homeostasis / lysosome / endosome membrane / endosome / lysosomal membrane
Similarity search - Function
Endosomal/lysomomal potassium channel TMEM175 / Endosomal/lysosomal potassium channel TMEM175
Similarity search - Domain/homology
: / Endosomal/lysosomal proton channel TMEM175
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsOh, S. / Paknejad, N. / Hite, R.K.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)P30 CA008748 United States
CitationJournal: Elife / Year: 2020
Title: Gating and selectivity mechanisms for the lysosomal K channel TMEM175.
Authors: SeCheol Oh / Navid Paknejad / Richard K Hite /
Abstract: Transmembrane protein 175 (TMEM175) is a K-selective ion channel expressed in lysosomal membranes, where it establishes a membrane potential essential for lysosomal function and its dysregulation is ...Transmembrane protein 175 (TMEM175) is a K-selective ion channel expressed in lysosomal membranes, where it establishes a membrane potential essential for lysosomal function and its dysregulation is associated with the development of Parkinson's Disease. TMEM175 is evolutionarily distinct from all known channels, predicting novel ion-selectivity and gating mechanisms. Here we present cryo-EM structures of human TMEM175 in open and closed conformations, enabled by resolutions up to 2.6 Å. Human TMEM175 adopts a homodimeric architecture with a central ion-conduction pore lined by the side chains of the pore-lining helices. Conserved isoleucine residues in the center of the pore serve as the gate in the closed conformation. In the widened channel in the open conformation, these same residues establish a constriction essential for K selectivity. These studies reveal the mechanisms of permeation, selectivity and gating and lay the groundwork for understanding the role of TMEM175 in lysosomal function.
History
DepositionMar 30, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 15, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 7, 2020Group: Database references / Category: citation / Item: _citation.title
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Endosomal/lysosomal potassium channel TMEM175
B: Endosomal/lysosomal potassium channel TMEM175
hetero molecules


Theoretical massNumber of molelcules
Total (without water)112,2659
Polymers111,3342
Non-polymers9307
Water72140
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8380 Å2
ΔGint-376 kcal/mol
Surface area33060 Å2

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Components

#1: Protein Endosomal/lysosomal potassium channel TMEM175 / Transmembrane protein 175 / hTMEM175


Mass: 55667.219 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TMEM175 / Production host: Homo sapiens (human) / References: UniProt: Q9BSA9
#2: Chemical
ChemComp-CS / CESIUM ION


Mass: 132.905 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Cs / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 40 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: Human open state TMEM175 in CsCl / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
10.1 %lauryl maltose neopentyl glycol1
250 mMTris pH 8.01
3150 mMCesium ChlorideCsCl1
42 mMDithiothreitol1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Dimeric channels
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: Blot for 4 seconds prior to plunging

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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 FIELD / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingAverage exposure time: 8 sec. / Electron dose: 61 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2SerialEMimage acquisition
4CTFFIND4.1.10CTF correction
7PHENIXdev-3751model fitting
9PHENIXdev-3751model refinement
10cryoSPARC2.12initial Euler assignment
11cryoSPARC2.12final Euler assignment
12cryoSPARC2.12classification
13cryoSPARC2.123D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4275219
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94653 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: FSC

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