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- EMDB-8881: Cryo-EM structure of mammalian endolysosomal TRPML1 channel in na... -

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Basic information

Entry
Database: EMDB / ID: EMD-8881
TitleCryo-EM structure of mammalian endolysosomal TRPML1 channel in nanodiscs in closed I conformation at 3.64 Angstrom resolution
Map dataCryo-EM structure of mammalian endolysosomal TRPML1 channel in nanodiscs in closed I conformation at 3.64 Angstrom resolution
Sample
  • Organelle or cellular component: Homotetramer of mouse TRPML1
    • Protein or peptide: Mucolipin-1
  • Ligand: SODIUM ION
KeywordsIon channel / MEMBRANE PROTEIN
Function / homology
Function and homology information


Transferrin endocytosis and recycling / positive regulation of lysosome organization / calcium ion export / intracellularly phosphatidylinositol-3,5-bisphosphate-gated monatomic cation channel activity / phagosome maturation / NAADP-sensitive calcium-release channel activity / iron ion transmembrane transporter activity / cellular response to pH / iron ion transmembrane transport / TRP channels ...Transferrin endocytosis and recycling / positive regulation of lysosome organization / calcium ion export / intracellularly phosphatidylinositol-3,5-bisphosphate-gated monatomic cation channel activity / phagosome maturation / NAADP-sensitive calcium-release channel activity / iron ion transmembrane transporter activity / cellular response to pH / iron ion transmembrane transport / TRP channels / monoatomic anion channel activity / endosomal transport / intracellular vesicle / sodium channel activity / monoatomic cation transmembrane transport / autophagosome maturation / potassium channel activity / phagocytic cup / monoatomic cation channel activity / release of sequestered calcium ion into cytosol / cellular response to calcium ion / cell projection / calcium channel activity / phagocytic vesicle membrane / late endosome membrane / late endosome / protein homotetramerization / adaptive immune response / lysosome / receptor complex / lysosomal membrane / lipid binding / Golgi apparatus / nucleoplasm / identical protein binding / membrane
Similarity search - Function
: / Mucolipin / : / Mucolipin, extracytosolic domain / Polycystin cation channel, PKD1/PKD2 / Polycystin cation channel
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.64 Å
AuthorsChen Q / She J
Funding support United States, 7 items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)GM079179 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)NS062792 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)AR060837 United States
Howard Hughes Medical Institute (HHMI) United States
Cancer Prevention and Research Institute of Texas (CPRIT) United States
Murchison Linthicum Scholar in Medical Research fund United States
Welch FoundationI-1578 United States
CitationJournal: Nature / Year: 2017
Title: Structure of mammalian endolysosomal TRPML1 channel in nanodiscs.
Authors: Qingfeng Chen / Ji She / Weizhong Zeng / Jiangtao Guo / Haoxing Xu / Xiao-Chen Bai / Youxing Jiang /
Abstract: Transient receptor potential mucolipin 1 (TRPML1) is a cation channel located within endosomal and lysosomal membranes. Ubiquitously expressed in mammalian cells, its loss-of-function mutations are ...Transient receptor potential mucolipin 1 (TRPML1) is a cation channel located within endosomal and lysosomal membranes. Ubiquitously expressed in mammalian cells, its loss-of-function mutations are the direct cause of type IV mucolipidosis, an autosomal recessive lysosomal storage disease. Here we present the single-particle electron cryo-microscopy structure of the mouse TRPML1 channel embedded in nanodiscs. Combined with mutagenesis analysis, the TRPML1 structure reveals that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P) binds to the N terminus of the channel-distal from the pore-and the helix-turn-helix extension between segments S2 and S3 probably couples ligand binding to pore opening. The tightly packed selectivity filter contains multiple ion-binding sites, and the conserved acidic residues form the luminal Ca-blocking site that confers luminal pH and Ca modulation on channel conductance. A luminal linker domain forms a fenestrated canopy atop the channel, providing several luminal ion passages to the pore and creating a negative electrostatic trap, with a preference for divalent cations, at the luminal entrance. The structure also reveals two equally distributed S4-S5 linker conformations in the closed channel, suggesting an S4-S5 linker-mediated PtdInsP gating mechanism among TRPML channels.
History
DepositionAug 7, 2017-
Header (metadata) releaseAug 30, 2017-
Map releaseOct 18, 2017-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.043
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.043
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5wpq
  • Surface level: 0.043
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8881.png

Downloads & links

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Map

FileDownload / File: emd_8881.map.gz / Format: CCP4 / Size: 29.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of mammalian endolysosomal TRPML1 channel in nanodiscs in closed I conformation at 3.64 Angstrom resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 198 pix.
= 211.86 Å		1.07 Å/pix.
x 198 pix.
= 211.86 Å		1.07 Å/pix.
x 198 pix.
= 211.86 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.043
Minimum - Maximum-0.12059494 - 0.1818826
Average (Standard dev.)0.0000108706145 (±0.011606903)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions198198198
Spacing198198198
CellA=B=C: 211.86002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z198198198
origin x/y/z0.0000.0000.000
length x/y/z211.860211.860211.860
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS198198198
D min/max/mean-0.1210.1820.000

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Supplemental data

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Sample components

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Entire : Homotetramer of mouse TRPML1

EntireName: Homotetramer of mouse TRPML1
Components
  • Organelle or cellular component: Homotetramer of mouse TRPML1
    • Protein or peptide: Mucolipin-1
  • Ligand: SODIUM ION

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Supramolecule #1: Homotetramer of mouse TRPML1

SupramoleculeName: Homotetramer of mouse TRPML1 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 66 KDa

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Macromolecule #1: Mucolipin-1

MacromoleculeName: Mucolipin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 66.656812 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MATPAGRRAS ETERLLTPNP GYGTQVGTSP APTTPTEEED LRRRLKYFFM SPCDKFRAKG RKPCKLMLQV VKILVVTVQL ILFGLSNQL VVTFREENTI AFRHLFLLGY SDGSDDTFAA YTQEQLYQAI FYAVDQYLIL PEISLGRYAY VRGGGGPWAN G SALALCQR ...String:
MATPAGRRAS ETERLLTPNP GYGTQVGTSP APTTPTEEED LRRRLKYFFM SPCDKFRAKG RKPCKLMLQV VKILVVTVQL ILFGLSNQL VVTFREENTI AFRHLFLLGY SDGSDDTFAA YTQEQLYQAI FYAVDQYLIL PEISLGRYAY VRGGGGPWAN G SALALCQR YYHRGHVDPA NDTFDIDPRV VTDCIQVDPP DRPPDIPSED LDFLDGSASY KNLTLKFHKL INVTIHFQLK TI NLQSLIN NEIPDCYTFS ILITFDNKAH SGRIPIRLET KTHIQECKHP SVSRHGDNSF RLLFDVVVIL TCSLSFLLCA RSL LRGFLL QNEFVVFMWR RRGREISLWE RLEFVNGWYI LLVTSDVLTI SGTVMKIGIE AKNLASYDVC SILLGTSTLL VWVG VIRYL TFFHKYNILI ATLRVALPSV MRFCCCVAVI YLGYCFCGWI VLGPYHVKFR SLSMVSECLF SLINGDDMFV TFAAM QAQQ GHSSLVWLFS QLYLYSFISL FIYMVLSLFI ALITGAYDTI KHPGGTGTEK SELQAYIEQC QDSPTSGKFR RGSGSA CSL FCCCGRDSPE DHSLLVNVDG GSSGGLVPR

UniProtKB: Mucolipin-1

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Macromolecule #3: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 3 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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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

Concentration1.3 mg/mL
BufferpH: 8 / Component - Concentration: 150.0 mM / Component - Formula: NaCl / Component - Name: sodium chloride
Details: Solutions were made fresh from stock solutions and filtered.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIFQuantum / Energy filter - Lower energy threshold: -10 eV / Energy filter - Upper energy threshold: 10 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3000 / Average exposure time: 15.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 46730
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Details30 frames per movie stack were saved for motion correction.
Particle selectionNumber selected: 1433949 / Details: The particles were auto-picked in RELION.
CTF correctionSoftware - Name: Gctf
Details: The CTF correction was performed during the map refinement in RELION.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: EMDB MAP
EMDB ID:

5778

Final reconstructionNumber classes used: 2 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2) / Number images used: 9000
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 2) / Details: The initial model is from a model of TRPV1.
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2)
Details: Auto-refinement in RELION generated the final reconstruction. The angular sampling was determined automatically in RELION.
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 2)
Details: We did the focus classification with density subtraction for the TM domain with RELION.

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