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- PDB-6hrr: Structure of the TRPML2 ELD at pH 6.5 -

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Basic information

Entry
Database: PDB / ID: 6hrr
TitleStructure of the TRPML2 ELD at pH 6.5
ComponentsMucolipin-2
KeywordsMEMBRANE PROTEIN / TRPML Channel Mucolipin Cation Channel Endolysosomal System
Function / homology
Function and homology information


positive regulation of macrophage inflammatory protein 1 alpha production / macrophage migration / NAADP-sensitive calcium-release channel activity / positive regulation of chemokine (C-C motif) ligand 5 production / iron ion transmembrane transporter activity / neutrophil migration / positive regulation of monocyte chemotactic protein-1 production / positive regulation of chemokine (C-X-C motif) ligand 2 production / TRP channels / calcium ion transmembrane transport ...positive regulation of macrophage inflammatory protein 1 alpha production / macrophage migration / NAADP-sensitive calcium-release channel activity / positive regulation of chemokine (C-C motif) ligand 5 production / iron ion transmembrane transporter activity / neutrophil migration / positive regulation of monocyte chemotactic protein-1 production / positive regulation of chemokine (C-X-C motif) ligand 2 production / TRP channels / calcium ion transmembrane transport / calcium channel activity / recycling endosome membrane / late endosome membrane / protein transport / adaptive immune response / lysosome / innate immune response / 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 speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2 Å
AuthorsBader, N. / Viet, K.K. / Wagner, A. / Hellmich, U.A.
CitationJournal: Structure / Year: 2019
Title: Structure of the Human TRPML2 Ion Channel Extracytosolic/Lumenal Domain.
Authors: Kerstin K Viet / Annika Wagner / Kevin Schwickert / Nils Hellwig / Martha Brennich / Nicole Bader / Tanja Schirmeister / Nina Morgner / Hermann Schindelin / Ute A Hellmich /
Abstract: TRPML2 is the least structurally characterized mammalian transient receptor potential mucolipin ion channel. The TRPML family hallmark is a large extracytosolic/lumenal domain (ELD) between ...TRPML2 is the least structurally characterized mammalian transient receptor potential mucolipin ion channel. The TRPML family hallmark is a large extracytosolic/lumenal domain (ELD) between transmembrane helices S1 and S2. We present crystal structures of the tetrameric human TRPML2 ELD at pH 6.5 (2.0 Å) and 4.5 (2.95 Å), corresponding to the pH values in recycling endosomes and lysosomes. Isothermal titration calorimetry shows Ca binding to the highly acidic central pre-pore loop which is abrogated at low pH, in line with a pH-dependent channel regulation model. Small angle X-ray scattering confirms the ELD dimensions in solution. Changes in pH or Ca concentration do not affect the protein's secondary structure, but can influence ELD oligomer integrity according to native mass spectrometry. Our data thus complete the set of high-resolution views of human TRPML channel ELDs and reveal some structural responses to the conditions the TRPML2 ELD encounters as the channel traffics through the endolysosomal system.
History
DepositionSep 28, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 19, 2019Provider: repository / Type: Initial release
Revision 1.1Aug 14, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 24, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_symmetry
Revision 1.3Oct 23, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Mucolipin-2
B: Mucolipin-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,17020
Polymers44,4262
Non-polymers1,74418
Water3,711206
1
A: Mucolipin-2
hetero molecules

A: Mucolipin-2
hetero molecules

A: Mucolipin-2
hetero molecules

A: Mucolipin-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)91,91440
Polymers88,8514
Non-polymers3,06336
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
crystal symmetry operation3_555-y,x,z1
crystal symmetry operation4_555y,-x,z1
Buried area11910 Å2
ΔGint-126 kcal/mol
Surface area34160 Å2
MethodPISA
2
B: Mucolipin-2
hetero molecules

B: Mucolipin-2
hetero molecules

B: Mucolipin-2
hetero molecules

B: Mucolipin-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)92,76540
Polymers88,8514
Non-polymers3,91436
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_465-x-1,-y+1,z1
crystal symmetry operation3_565-y,x+1,z1
crystal symmetry operation4_455y-1,-x,z1
Buried area13500 Å2
ΔGint-68 kcal/mol
Surface area34420 Å2
MethodPISA
Unit cell
Length a, b, c (Å)109.471, 109.471, 149.744
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number97
Space group name H-MI422
Components on special symmetry positions
IDModelComponents
11A-301-

K

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Mucolipin-2 / Transient receptor potential channel mucolipin 2 / TRPML2


Mass: 22212.836 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCOLN2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8IZK6

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Non-polymers , 5 types, 224 molecules

#2: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#4: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID


Mass: 195.237 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 206 / Source method: isolated from a natural source / Formula: H2O

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Details

Has protein modificationY

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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: 2.52 Å3/Da / Density % sol: 51.28 % / Description: Cube
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5 / Details: 30% Jeffamine M-600 0.1 M MES pH 6.5 50 mM CsCl

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID30B / Wavelength: 0.9677 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: May 11, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9677 Å / Relative weight: 1
ReflectionResolution: 2→46.53 Å / Num. obs: 31030 / % possible obs: 99.9 % / Redundancy: 27.2 % / Biso Wilson estimate: 47.23 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.102 / Rpim(I) all: 0.002 / Net I/σ(I): 21.5
Reflection shellResolution: 2→2.05 Å / Redundancy: 28.8 % / Rmerge(I) obs: 3.556 / Mean I/σ(I) obs: 1.3 / Num. unique obs: 2240 / CC1/2: 0.645 / Rpim(I) all: 0.673 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
XDSJan 26, 2018data reduction
Aimless0.7.2data scaling
PHASER2.8.2phasing
BUSTER2.10.3refinement
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5TJA

5tja


Resolution: 2→20 Å / Cor.coef. Fo:Fc: 0.952 / Cor.coef. Fo:Fc free: 0.943 / SU R Cruickshank DPI: 0.263 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.166 / SU Rfree Blow DPI: 0.143 / SU Rfree Cruickshank DPI: 0.141
RfactorNum. reflection% reflectionSelection details
Rfree0.217 1560 5.03 %RANDOM
Rwork0.186 ---
obs0.188 30988 99.9 %-
Displacement parametersBiso max: 202.68 Å2 / Biso mean: 61.21 Å2 / Biso min: 23.95 Å2
Baniso -1Baniso -2Baniso -3
1-5.5228 Å20 Å20 Å2
2--5.5228 Å20 Å2
3----11.0455 Å2
Refine analyzeLuzzati coordinate error obs: 0.24 Å
Refinement stepCycle: final / Resolution: 2→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2841 0 106 212 3159
Biso mean--84.06 60.66 -
Num. residues----346
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d1715SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes
X-RAY DIFFRACTIONt_gen_planes989HARMONIC5
X-RAY DIFFRACTIONt_it5971HARMONIC20
X-RAY DIFFRACTIONt_nbd0SEMIHARMONIC5
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion386SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance4HARMONIC1
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact6327SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d5971HARMONIC20.01
X-RAY DIFFRACTIONt_angle_deg10713HARMONIC21.02
X-RAY DIFFRACTIONt_omega_torsion3.69
X-RAY DIFFRACTIONt_other_torsion3.07
LS refinement shellResolution: 2→2.07 Å / Rfactor Rfree error: 0 / Total num. of bins used: 16
RfactorNum. reflection% reflection
Rfree0.2486 152 5.39 %
Rwork0.2214 2669 -
all0.2229 2821 -
obs--100 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.6966-0.5502-0.50681.66210.19751.9923-0.0141-0.11820.2281-0.00190.0450.0751-0.0594-0.0601-0.031-0.16930.0347-0.0032-0.1590.0027-0.1744-15.66920.7254-14.1139
23.0913-0.1158-0.23962.2564-0.29691.2179-0.0772-0.096-0.42760.03230.0143-0.18330.14990.0560.0629-0.19060.03270.001-0.21730.0433-0.0957-39.878433.8884-27.2479
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|* }A92 - 282
2X-RAY DIFFRACTION2{ B|* }B92 - 282

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