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- PDB-5wpt: Cryo-EM structure of mammalian endolysosomal TRPML1 channel in na... -

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Basic information

Entry
Database: PDB / ID: 5wpt
TitleCryo-EM structure of mammalian endolysosomal TRPML1 channel in nanodiscs in closed II conformation at 3.75 Angstrom resolution
ComponentsMucolipin-1
KeywordsMEMBRANE PROTEIN / Ion channel
Function / homology
Function and homology information


Transferrin endocytosis and recycling / calcium ion export / positive regulation of lysosome organization / intracellularly phosphatidylinositol-3,5-bisphosphate-gated monatomic cation channel activity / phagosome maturation / NAADP-sensitive calcium-release channel activity / iron ion transmembrane transporter activity / iron ion transmembrane transport / cellular response to pH / monoatomic anion channel activity ...Transferrin endocytosis and recycling / calcium ion export / positive regulation of lysosome organization / intracellularly phosphatidylinositol-3,5-bisphosphate-gated monatomic cation channel activity / phagosome maturation / NAADP-sensitive calcium-release channel activity / iron ion transmembrane transporter activity / iron ion transmembrane transport / cellular response to pH / monoatomic anion channel activity / TRP channels / sodium channel activity / endosomal transport / intracellular vesicle / monoatomic cation transmembrane transport / phagocytic cup / autophagosome maturation / potassium channel activity / 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 / late endosome membrane / protein homotetramerization / adaptive immune response / lysosome / receptor complex / lysosomal membrane / lipid binding / Golgi apparatus / nucleoplasm / identical protein binding / membrane / plasma 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsChen, Q. / She, J. / Guo, J. / Bai, X. / Jiang, Y.
Funding support United States, 7items
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, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 18, 2017Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2017Group: Database references / Category: citation / citation_author
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Apr 24, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Apr 5, 2023Group: Database references / Structure summary / Category: chem_comp / database_2 / pdbx_database_related
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 2.2Oct 16, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Mucolipin-1
B: Mucolipin-1
C: Mucolipin-1
D: Mucolipin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)268,3489
Polymers266,6274
Non-polymers1,7215
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area28770 Å2
ΔGint-207 kcal/mol
Surface area93930 Å2

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Components

#1: Protein
Mucolipin-1 / TRPML1 / Mucolipidin


Mass: 66656.812 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Mcoln1 / Plasmid: pEZT / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q99J21
#2: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
Has protein modificationY

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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: Homotetramer of mouse TRPML1 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.066 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F / Plasmid: pEZT
Buffer solutionpH: 8
Details: Solutions were made fresh from stock solutions and filtered.
Buffer componentConc.: 150 mM / Name: sodium chloride / Formula: NaCl
SpecimenConc.: 1.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 FIELD / Nominal magnification: 46730 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3000
EM imaging opticsEnergyfilter name: GIFQuantum / Energyfilter upper: 10 eV / Energyfilter lower: -10 eV
Image scansMovie frames/image: 30

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2particle selection
2EPUimage acquisition
4GctfCTF correction
10RELION2initial Euler assignment
11RELION2final Euler assignment
12RELION2classification
13RELION23D reconstruction
Image processingDetails: 30 frames per movie stack were saved for motion correction.
CTF correctionDetails: The CTF correction was performed during the map refinement in RELION.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1433949 / Details: The particles were auto-picked in RELION.
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11000 / Algorithm: BACK PROJECTION / Num. of class averages: 3 / Symmetry type: 2D CRYSTAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00815224
ELECTRON MICROSCOPYf_angle_d1.03320652
ELECTRON MICROSCOPYf_dihedral_angle_d12.5198920
ELECTRON MICROSCOPYf_chiral_restr0.0542420
ELECTRON MICROSCOPYf_plane_restr0.0082516

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