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- PDB-5tjc: I-II linker of TRPML1 channel at pH 7.5 -

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Basic information

Entry
Database: PDB / ID: 5tjc
TitleI-II linker of TRPML1 channel at pH 7.5
ComponentsMucolipin-1MCOLN1
KeywordsTRANSPORT PROTEIN / endolysosomal lumen / tetramer / calcium and pH regulation
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 / phagocytic cup / autophagosome maturation / monoatomic cation transport / localization / monoatomic cation channel activity / release of sequestered calcium ion into cytosol / 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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.4 Å
AuthorsLi, M. / Zhang, W.K. / Benvin, N.M. / Zhou, X. / Su, D. / Li, H. / Wang, S. / Michailidis, I.E. / Tong, L. / Li, X. / Yang, J.
Funding support United States, China, 8items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM085234 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)RO1NS053494 United States
National Basic Research Program of China (973 Program)2014CB910301 China
National Natural Science Foundation of China (NSFC)31370821 China
Top Talents Program of Yunnan Province2011HA012 China
High-level Overseas Talents of Yunnan Province China
China Youth 1000-Talent Program of the State Council of China China
Beijing Advanced Innovation Center for Structural Biology China
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: Structural basis of dual Ca/pH regulation of the endolysosomal TRPML1 channel.
Authors: Minghui Li / Wei K Zhang / Nicole M Benvin / Xiaoyuan Zhou / Deyuan Su / Huan Li / Shu Wang / Ioannis E Michailidis / Liang Tong / Xueming Li / Jian Yang /
Abstract: The activities of organellar ion channels are often regulated by Ca and H, which are present in high concentrations in many organelles. Here we report a structural element critical for dual Ca/pH ...The activities of organellar ion channels are often regulated by Ca and H, which are present in high concentrations in many organelles. Here we report a structural element critical for dual Ca/pH regulation of TRPML1, a Ca-release channel crucial for endolysosomal function. TRPML1 mutations cause mucolipidosis type IV (MLIV), a severe lysosomal storage disorder characterized by neurodegeneration, mental retardation and blindness. We obtained crystal structures of the 213-residue luminal domain of human TRPML1 containing three missense MLIV-causing mutations. This domain forms a tetramer with a highly electronegative central pore formed by a novel luminal pore loop. Cysteine cross-linking and cryo-EM analyses confirmed that this architecture occurs in the full-length channel. Structure-function studies demonstrated that Ca and H interact with the luminal pore and exert physiologically important regulation. The MLIV-causing mutations disrupt the luminal-domain structure and cause TRPML1 mislocalization. Our study reveals the structural underpinnings of TRPML1's regulation, assembly and pathogenesis.
History
DepositionOct 4, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 25, 2017Provider: repository / Type: Initial release
Revision 1.1Feb 8, 2017Group: Database references
Revision 1.2Mar 15, 2017Group: Database references
Revision 1.3Sep 27, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Oct 4, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Mucolipin-1


Theoretical massNumber of molelcules
Total (without water)25,1951
Polymers25,1951
Non-polymers00
Water1,18966
1
A: Mucolipin-1

A: Mucolipin-1

A: Mucolipin-1

A: Mucolipin-1


Theoretical massNumber of molelcules
Total (without water)100,7804
Polymers100,7804
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
crystal symmetry operation15_555y,-x,z1
crystal symmetry operation16_555-y,x,z1
Buried area11060 Å2
ΔGint-75 kcal/mol
Surface area32310 Å2
MethodPISA
Unit cell
Length a, b, c (Å)182.811, 182.811, 182.811
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number209
Space group name H-MF432

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Components

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


Mass: 25195.057 Da / Num. of mol.: 1 / Fragment: UNP residues (84-296)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCOLN1, ML4, MSTP080 / Plasmid: pET26b / Production host: Escherichia coli (E. coli) / Strain (production host): DE3 / Variant (production host): Rosetta-gami 2 / References: UniProt: Q9GZU1
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 66 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.53 Å3/Da / Density % sol: 51.3 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 100 mM magnesium sulfate, 4% PEG 3350, 100 mM HEPES at pH 7.5, macroseeding

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X29A / Wavelength: 1.075 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Nov 19, 2012
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.075 Å / Relative weight: 1
ReflectionResolution: 2.4→20 Å / Num. obs: 10548 / % possible obs: 98 % / Redundancy: 4.2 % / Rsym value: 0.059 / Net I/σ(I): 22.3
Reflection shellResolution: 2.4→2.49 Å / Redundancy: 4.1 % / Rmerge(I) obs: 0.34 / Mean I/σ(I) obs: 3.7 / % possible all: 98.6

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHASERphasing
CNSrefinement
PDB_EXTRACT3.2data extraction
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5TJA

5tja


Resolution: 2.4→20 Å / Cross valid method: THROUGHOUT / σ(F): 0
RfactorNum. reflection% reflectionSelection details
Rfree0.2533 1033 9.6 %Random selection
Rwork0.2156 ---
obs-10121 94.3 %-
Solvent computationBsol: 47.6387 Å2
Displacement parametersBiso max: 112.79 Å2 / Biso mean: 50.4821 Å2 / Biso min: 24.47 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å20 Å2
3---0 Å2
Refinement stepCycle: final / Resolution: 2.4→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1426 0 66 0 1492
Biso mean--47.81 --
Num. residues----176
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.006
X-RAY DIFFRACTIONc_angle_d1.385
X-RAY DIFFRACTIONc_mcbond_it1.6641.5
X-RAY DIFFRACTIONc_scbond_it2.1892
X-RAY DIFFRACTIONc_mcangle_it2.9342
X-RAY DIFFRACTIONc_scangle_it3.3332.5
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 10

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.4-2.490.3282980.276883092888.5
2.49-2.580.286870.252385193890.4
2.58-2.70.3195810.270887395491.8
2.7-2.840.32141000.279489399394
2.84-3.020.2573990.229589999896.2
3.02-3.250.27651280.2143901102996.6
3.25-3.580.23661060.2038939104598
3.58-4.090.241120.2042940105297.3
4.09-5.140.23971090.1803960106996.8
5.14-200.22281130.21711002111593
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1CNS_TOPPAR:protein_rep.paramCNS_TOPPAR:protein.top
X-RAY DIFFRACTION2CNS_TOPPAR:dna-rna_rep.paramCNS_TOPPAR:dna-rna.top
X-RAY DIFFRACTION3CNS_TOPPAR:water_rep.paramCNS_TOPPAR:water.top
X-RAY DIFFRACTION4CNS_TOPPAR:ion.paramCNS_TOPPAR:ion.top
X-RAY DIFFRACTION5CNS_TOPPAR:carbohydrate.paramCNS_TOPPAR:carbohydrate.top

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