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- PDB-5wj5: Human TRPML1 channel structure in closed conformation -

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Basic information

Entry
Database: PDB / ID: 5wj5
TitleHuman TRPML1 channel structure in closed conformation
ComponentsMucolipin-1MCOLN1
KeywordsMEMBRANE PROTEIN / human TRPML1
Function / homology
Function and homology information


calcium ion export / positive regulation of lysosome organization / iron ion transmembrane transporter activity / intracellularly phosphatidylinositol-3,5-bisphosphate-gated monatomic cation channel activity / NAADP-sensitive calcium-release channel activity / phagosome maturation / transferrin transport / Transferrin endocytosis and recycling / cellular response to pH / ligand-gated calcium channel activity ...calcium ion export / positive regulation of lysosome organization / iron ion transmembrane transporter activity / intracellularly phosphatidylinositol-3,5-bisphosphate-gated monatomic cation channel activity / NAADP-sensitive calcium-release channel activity / phagosome maturation / transferrin transport / Transferrin endocytosis and recycling / cellular response to pH / ligand-gated calcium channel activity / TRP channels / autophagosome maturation / monoatomic cation transport / phagocytic cup / localization / monoatomic cation channel activity / cellular response to calcium ion / cell projection / calcium ion transmembrane transport / calcium channel activity / phagocytic vesicle membrane / late endosome / late endosome membrane / protein homotetramerization / adaptive immune response / lysosome / receptor complex / endosome membrane / lysosomal membrane / intracellular membrane-bounded organelle / lipid binding / Golgi apparatus / nucleoplasm / membrane / identical protein binding / plasma membrane
Similarity search - Function
: / : / Mucolipin, extracytosolic domain / Mucolipin / Polycystin cation channel, PKD1/PKD2 / Polycystin cation channel
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.72 Å
AuthorsSchmiege, P. / Li, X.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Endowed Scholars Program in Medical Science of UT southwestern medical center United States
CitationJournal: Nature / Year: 2017
Title: Human TRPML1 channel structures in open and closed conformations.
Authors: Philip Schmiege / Michael Fine / Günter Blobel / Xiaochun Li /
Abstract: Transient receptor potential mucolipin 1 (TRPML1) is a Ca-releasing cation channel that mediates the calcium signalling and homeostasis of lysosomes. Mutations in TRPML1 lead to mucolipidosis type ...Transient receptor potential mucolipin 1 (TRPML1) is a Ca-releasing cation channel that mediates the calcium signalling and homeostasis of lysosomes. Mutations in TRPML1 lead to mucolipidosis type IV, a severe lysosomal storage disorder. Here we report two electron cryo-microscopy structures of full-length human TRPML1: a 3.72-Å apo structure at pH 7.0 in the closed state, and a 3.49-Å agonist-bound structure at pH 6.0 in an open state. Several aromatic and hydrophobic residues in pore helix 1, helices S5 and S6, and helix S6 of a neighbouring subunit, form a hydrophobic cavity to house the agonist, suggesting a distinct agonist-binding site from that found in TRPV1, a TRP channel from a different subfamily. The opening of TRPML1 is associated with distinct dilations of its lower gate together with a slight structural movement of pore helix 1. Our work reveals the regulatory mechanism of TRPML channels, facilitates better understanding of TRP channel activation, and provides insights into the molecular basis of mucolipidosis type IV pathogenesis.
History
DepositionJul 21, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 18, 2017Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2017Group: Database references / Structure summary / Category: citation / struct
Item: _citation.pdbx_database_id_PubMed / _citation.title / _struct.title
Revision 1.2Nov 1, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Nov 8, 2017Group: Derived calculations / Category: pdbx_struct_assembly
Item: _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details
Revision 1.4Oct 3, 2018Group: Data collection / Other / Refinement description / Category: cell / refine
Item: _cell.Z_PDB / _cell.length_a ..._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
Revision 1.6Dec 18, 2019Group: Other / Category: atom_sites
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]

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Structure visualization

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Structure viewerMolecule:
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)260,3404
Polymers260,3404
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area29020 Å2
ΔGint-261 kcal/mol
Surface area89630 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14B
24C
15B
25D
16C
26D

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A60 - 526
2010B60 - 526
1020A60 - 526
2020C60 - 526
1030A60 - 526
2030D60 - 526
1040B60 - 526
2040C60 - 526
1050B60 - 526
2050D60 - 526
1060C60 - 526
2060D60 - 526

NCS ensembles :
ID
1
2
3
4
5
6

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Components

#1: Protein
Mucolipin-1 / MCOLN1 / MG-2 / Mucolipidin


Mass: 65084.996 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCOLN1, ML4, MSTP080 / Cell line (production host): HEK-293S / Production host: Homo sapiens (human) / References: UniProt: Q9GZU1

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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: TRPML1MCOLN1 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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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 recordingElectron dose: 1.6 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0158 / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
4CTFFIND4CTF correction
13FREALIGN3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56105 / Symmetry type: POINT
RefinementResolution: 3.7→78.69 Å / Cor.coef. Fo:Fc: 0.956 / SU B: 50.279 / SU ML: 0.668
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.30408 --
obs0.30408 55348 99.99 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 272.154 Å2
Baniso -1Baniso -2Baniso -3
1--0.07 Å20.03 Å20.25 Å2
2---0.07 Å20.17 Å2
3---0.14 Å2
Refinement stepCycle: 1 / Total: 14640
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.01915022
ELECTRON MICROSCOPYr_bond_other_d0.0020.0214068
ELECTRON MICROSCOPYr_angle_refined_deg1.2531.95220434
ELECTRON MICROSCOPYr_angle_other_deg0.961332406
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.68951818
ELECTRON MICROSCOPYr_dihedral_angle_2_deg33.39422.761652
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.827152464
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.2371592
ELECTRON MICROSCOPYr_chiral_restr0.0710.22352
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.0216474
ELECTRON MICROSCOPYr_gen_planes_other0.0010.023346
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_it13.0427.9237302
ELECTRON MICROSCOPYr_mcbond_other13.03327.9237301
ELECTRON MICROSCOPYr_mcangle_it21.25541.8279110
ELECTRON MICROSCOPYr_mcangle_other21.25541.8279111
ELECTRON MICROSCOPYr_scbond_it11.26427.967720
ELECTRON MICROSCOPYr_scbond_other11.26327.9617721
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other19.41141.87811325
ELECTRON MICROSCOPYr_long_range_B_refined32.48718545
ELECTRON MICROSCOPYr_long_range_B_other32.48618546
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A284680.08
12B284680.08
21A284700.08
22C284700.08
31A284860.08
32D284860.08
41B285860.07
42C285860.07
51B285300.07
52D285300.07
61C285300.07
62D285300.07
LS refinement shellResolution: 3.7→3.796 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.654 4061 -
Rfree-0 -
obs--99.95 %

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