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- EMDB-6670: The endolysosomal Ca2+ channel TRPML1 -

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Basic information

Entry
Database: EMDB / ID: EMD-6670
TitleThe endolysosomal Ca2+ channel TRPML1
Map dataNone
Sample
  • Complex: The endolysosomal Ca2+ channel TRPML1
    • Protein or peptide: The endolysosomal Ca2+ channel TRPML1
Biological speciesCaenorhabditis elegans (invertebrata)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.28 Å
AuthorsLi X / Zhou X
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
DepositionNov 15, 2016-
Header (metadata) releaseJan 11, 2017-
Map releaseJan 11, 2017-
UpdateSep 5, 2018-
Current statusSep 5, 2018Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0981
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0981
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6670.png

Downloads & links

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Map

FileDownload / File: emd_6670.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.64 Å/pix.
x 80 pix.
= 211.2 Å		2.64 Å/pix.
x 80 pix.
= 211.2 Å		2.64 Å/pix.
x 80 pix.
= 211.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.64 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0981 / Movie #1: 0.0981
Minimum - Maximum-0.20162292 - 0.44207188
Average (Standard dev.)0.0038177674 (±0.022139238)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 211.20001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.642.642.64
M x/y/z808080
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/NS808080
D min/max/mean-0.2020.4420.004

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Supplemental data

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Additional map: None

Fileemd_6670_additional.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The endolysosomal Ca2+ channel TRPML1

EntireName: The endolysosomal Ca2+ channel TRPML1
Components
  • Complex: The endolysosomal Ca2+ channel TRPML1
    • Protein or peptide: The endolysosomal Ca2+ channel TRPML1

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Supramolecule #1: The endolysosomal Ca2+ channel TRPML1

SupramoleculeName: The endolysosomal Ca2+ channel TRPML1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: pFastBac1

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Macromolecule #1: The endolysosomal Ca2+ channel TRPML1

MacromoleculeName: The endolysosomal Ca2+ channel TRPML1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GGGGSMSRRS TTDRSDFNDN ASNASSNASR RPTINFQEIM EDLPHHERET GERLRRHLQF FFMNPMEKWK VRHQLPYKLV LQVLKIVFVT MQLILFAEMR MSHVDFLEDT TTVMRHRFLK EWNDDRDALQ YPPAEGRYSV YDDQGLSEHL SFLINSYYSI RNDSFASFSY ...String:
GGGGSMSRRS TTDRSDFNDN ASNASSNASR RPTINFQEIM EDLPHHERET GERLRRHLQF FFMNPMEKWK VRHQLPYKLV LQVLKIVFVT MQLILFAEMR MSHVDFLEDT TTVMRHRFLK EWNDDRDALQ YPPAEGRYSV YDDQGLSEHL SFLINSYYSI RNDSFASFSY DVVSHPSGNL GAQISFESIP PIEVLIDRIS NVTVNNNTYN FDIREVKDTK RLNLTETEVF QIGQSDDAVR DILATRGITF LPEDALKIST VQFKFRLRTI HYSPTAGDQK PECYKISVSI KFDNSRHTGQ VHVTLSTVVS YVNVCNGRII KGVGWSFDTL LIGGTDIFVL ILCILSLILC CRALIKAHLL QIKTSDYFEN VLKNKITVTD QLDFLNLWYV MIVVNDALII IGTVAKISIE FQDFDNSLFT LTSIFLGMGA LLVYVGVLRY FGFFSQYNIL MLTLKRSAPN IMRFMTCAIV LYAGFLIAGW VIIGPYSMKF RTLAESSEAL FSLLNGDDMF ATFYTINDSN TVIKVFGTVY IYLFVSLFIY VVLSLFIAII MDAYEVVKDR YSDGLRAIEK RGCLRDFVES NPPPSELGSP TTRSAYAPSN LLNLGRGWQR LE

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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

Concentration0.6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMC8H18N2O4S4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
GridModel: R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV
Details: waiting for 3 seconds before blotting for 3.5 seconds(double-sided, blot force 1),then the grid was immediately plunged into liquid ethane cooled by liquid-nitrogen..
Detailsthe sample was homogeneous

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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 - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-32 / Average exposure time: 8.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 2.1 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: OTHER
Details: an four pieces of propeller was drawn manually as initial model
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 5.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 71052
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final 3D classificationSoftware - Name: RELION (ver. 1.4)

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