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- EMDB-7018: Human apo-TRPML3 channel at pH 7.4 -

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Basic information

Entry
Database: EMDB / ID: EMD-7018
TitleHuman apo-TRPML3 channel at pH 7.4
Map data
Sample
  • Complex: human TRPML3 apo channel at pH 7.4
    • Protein or peptide: Mucolipin-3
Keywordsion channel / TRP channel / lysosomal / TRANSPORT PROTEIN
Function / homology
Function and homology information


NAADP-sensitive calcium-release channel activity / inner ear auditory receptor cell differentiation / TRP channels / autophagosome membrane / locomotory behavior / calcium ion transmembrane transport / calcium channel activity / late endosome membrane / early endosome membrane / lysosomal membrane ...NAADP-sensitive calcium-release channel activity / inner ear auditory receptor cell differentiation / TRP channels / autophagosome membrane / locomotory behavior / calcium ion transmembrane transport / calcium channel activity / late endosome membrane / early endosome membrane / lysosomal membrane / lipid 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.06 Å
AuthorsZhou X / Li M
Funding support China, United States, 9 items
OrganizationGrant numberCountry
National Basic Research Program of China (973 Program)2014CB910301 China
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM085234 United States
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
Tsinghua-Peking Joint Center for Life Sciences China
National Natural Science Foundation of China (NSFC)31570730 China
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: Cryo-EM structures of the human endolysosomal TRPML3 channel in three distinct states.
Authors: Xiaoyuan Zhou / Minghui Li / Deyuan Su / Qi Jia / Huan Li / Xueming Li / Jian Yang /
Abstract: TRPML3 channels are mainly localized to endolysosomes and play a critical role in the endocytic pathway. Their dysfunction causes deafness and pigmentation defects in mice. TRPML3 activity is ...TRPML3 channels are mainly localized to endolysosomes and play a critical role in the endocytic pathway. Their dysfunction causes deafness and pigmentation defects in mice. TRPML3 activity is inhibited by low endolysosomal pH. Here we present cryo-electron microscopy (cryo-EM) structures of human TRPML3 in the closed, agonist-activated, and low-pH-inhibited states, with resolutions of 4.06, 3.62, and 4.65 Å, respectively. The agonist ML-SA1 lodges between S5 and S6 and opens an S6 gate. A polycystin-mucolipin domain (PMD) forms a luminal cap. S1 extends into this cap, forming a 'gating rod' that connects directly to a luminal pore loop, which undergoes dramatic conformational changes in response to low pH. S2 extends intracellularly and interacts with several intracellular regions to form a 'gating knob'. These unique structural features, combined with the results of electrophysiological studies, indicate a new mechanism by which luminal pH and other physiological modulators such as PIP regulate TRPML3 by changing S1 and S2 conformations.
History
DepositionSep 8, 2017-
Header (metadata) releaseNov 1, 2017-
Map releaseNov 8, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6aye
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_7018.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.45451486 - 0.5796037
Average (Standard dev.)0.0019367598 (±0.023540122)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 211.20001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z211.200211.200211.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.4550.5800.002

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Supplemental data

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Sample components

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Entire : human TRPML3 apo channel at pH 7.4

EntireName: human TRPML3 apo channel at pH 7.4
Components
  • Complex: human TRPML3 apo channel at pH 7.4
    • Protein or peptide: Mucolipin-3

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Supramolecule #1: human TRPML3 apo channel at pH 7.4

SupramoleculeName: human TRPML3 apo channel at pH 7.4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Mucolipin-3

MacromoleculeName: Mucolipin-3 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 64.625785 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GGGGSMADPE VVVSSCSSHE EENRCNFNQQ TSPSEELLLE DQMRRKLKFF FMNPCEKFWA RGRKPWKLAI QILKIAMVTI QLVLFGLSN QMVVAFKEEN TIAFKHLFLK GYMDRMDDTY AVYTQSDVYD QLIFAVNQYL QLYNVSVGNH AYENKGTKQS A MAICQHFY ...String:
GGGGSMADPE VVVSSCSSHE EENRCNFNQQ TSPSEELLLE DQMRRKLKFF FMNPCEKFWA RGRKPWKLAI QILKIAMVTI QLVLFGLSN QMVVAFKEEN TIAFKHLFLK GYMDRMDDTY AVYTQSDVYD QLIFAVNQYL QLYNVSVGNH AYENKGTKQS A MAICQHFY KRGNIYPGND TFDIDPEIET ECFFVEPDEP FHIGTPAENK LNLTLDFHRL LTVELQFKLK AINLQTVRHQ EL PDCYDFT LTITFDNKAH SGRIKISLDN DISIRECKDW HVSGSIQKNT HYMMIFDAFV ILTCLVSLIL CIRSVIRGLQ LQQ EFVNFF LLHYKKEVSV SDQMEFVNGW YIMIIISDIL TIIGSILKME IQAKSLTSYD VCSILLGTST MLVWLGVIRY LGFF AKYNL LILTLQAALP NVIRFCCCAA MIYLGYCFCG WIVLGPYHDK FRSLNMVSEC LFSLINGDDM FATFAKMQQK SYLVW LFSR IYLYSFISLF IYMILSLFIA LITDTYETIK QYQQDGFPET ELRTFISECK DLPNSGKYRL EDDPPVSLFC CCKK

UniProtKB: Mucolipin-3

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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

Concentration1.9 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride

Details: The pH of the buffer was adjusted to 7.4 by NaOH.
GridModel: Quantifoil holey carbon grid R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
Details: waiting for 3 seconds before blotting for 4 seconds(double-sided, blot force 1),then the grid was immediately plunged into liquid ethane cooled by liquid-nitrogen.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 1-32 / Average exposure time: 8.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final 3D classificationSoftware - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 43542

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