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- EMDB-7972: cryo-EM reconstruction of the mitochondrial calcium uniporter (MC... -

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Basic information

Entry
Database: EMDB / ID: EMD-7972
Titlecryo-EM reconstruction of the mitochondrial calcium uniporter (MCU) from Zebrafish
Map dataCryo-EM structure of the mitochondrial calcium uniporter
Sample
  • Organelle or cellular component: Mitochondrial Calcium Uniporter (MCU) from Zebrafish
    • Protein or peptide: Mitochondrial Calcium Uniporter (MCU) from Zebrafish
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.5 Å
AuthorsLong SB / Baradaran R / Wang C
CitationJournal: Nature / Year: 2018
Title: Cryo-EM structures of fungal and metazoan mitochondrial calcium uniporters.
Authors: Rozbeh Baradaran / Chongyuan Wang / Andrew Francis Siliciano / Stephen Barstow Long /
Abstract: The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel and a major route of calcium entry into mitochondria. How the channel catalyses ion permeation and achieves ...The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel and a major route of calcium entry into mitochondria. How the channel catalyses ion permeation and achieves ion selectivity are not well understood, partly because MCU is thought to have a distinct architecture in comparison to other cellular channels. Here we report cryo-electron microscopy reconstructions of MCU channels from zebrafish and Cyphellophora europaea at 8.5 Å and 3.2 Å resolutions, respectively. In contrast to a previous report of pentameric stoichiometry for MCU, both channels are tetramers. The atomic model of C. europaea MCU shows that a conserved WDXXEP signature sequence forms the selectivity filter, in which calcium ions are arranged in single file. Coiled-coil legs connect the pore to N-terminal domains in the mitochondrial matrix. In C. europaea MCU, the N-terminal domains assemble as a dimer of dimers; in zebrafish MCU, they form an asymmetric crescent. The structures define principles that underlie ion permeation and calcium selectivity in this unusual channel.
History
DepositionJun 6, 2018-
Header (metadata) releaseJun 27, 2018-
Map releaseJul 18, 2018-
UpdateAug 8, 2018-
Current statusAug 8, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.55
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.55
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7972.png

Downloads & links

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Map

FileDownload / File: emd_7972.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the mitochondrial calcium uniporter
Voxel sizeX=Y=Z: 1.384 Å
Density
Contour LevelBy AUTHOR: 1.55 / Movie #1: 1.55
Minimum - Maximum-2.6261976 - 10.533556000000001
Average (Standard dev.)-0.022866152 (±0.3109403)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 354.304 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3841.3841.384
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z354.304354.304354.304
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.62610.534-0.023

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Supplemental data

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Sample components

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Entire : Mitochondrial Calcium Uniporter (MCU) from Zebrafish

EntireName: Mitochondrial Calcium Uniporter (MCU) from Zebrafish
Components
  • Organelle or cellular component: Mitochondrial Calcium Uniporter (MCU) from Zebrafish
    • Protein or peptide: Mitochondrial Calcium Uniporter (MCU) from Zebrafish

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Supramolecule #1: Mitochondrial Calcium Uniporter (MCU) from Zebrafish

SupramoleculeName: Mitochondrial Calcium Uniporter (MCU) from Zebrafish / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 133 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293S GnTI -

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Macromolecule #1: Mitochondrial Calcium Uniporter (MCU) from Zebrafish

MacromoleculeName: Mitochondrial Calcium Uniporter (MCU) from Zebrafish / type: protein_or_peptide / ID: 1
Details: Zebrafish (Danio rerio) MCU expressed in mammalian cells and purified in digitonin detergent.
Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: LLCSPAAEDV SVVYQNGLPV ISVRLPSRRE R CQFTLKPL SDTVGVFLQQ LQAEDRGIDR VTIYSADGAR IASSTGIDIL LMDNFKLVIN DT SYLVQPP RRDLLPHEDG ERLNDVKILV QQLYTTLRIE EHQLNKEREL IGRLEDLNSQ LQP LEKVKE ELSKKAERRT ...String:
LLCSPAAEDV SVVYQNGLPV ISVRLPSRRE R CQFTLKPL SDTVGVFLQQ LQAEDRGIDR VTIYSADGAR IASSTGIDIL LMDNFKLVIN DT SYLVQPP RRDLLPHEDG ERLNDVKILV QQLYTTLRIE EHQLNKEREL IGRLEDLNSQ LQP LEKVKE ELSKKAERRT TWVLWGGMAY MATQFGILAR LTWWEYSWDI MEPVTYFITY GTAM AMYAY FVLTRQEYLY PDARDRQYLL FFHRGAKRTR FDIEKYNKLK DAIAEAELDL KRLRD PLQL NLPIQQIDTS KD

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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

Concentration5 mg/mL
BufferpH: 8.5
Component:
ConcentrationFormulaName
20.0 mMTrisTris(hydroxymethyl)-aminomethane
150.0 mMNaClSodium chloridesodium chloride
0.005 mMCaCl2calcium chloride
0.4 mMTCEPTris(2-carboxyethyl)phosphine
0.5 mMdigitonindigitonin
0.01 mg/mLcardiolipin1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 2 second blot, blot force of 0.
DetailsMonodisperse sample

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 37000
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 - Frames/image: 1-46 / Number grids imaged: 3 / Number real images: 3500 / Average exposure time: 0.13 sec. / Average electron dose: 2.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 269574
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.5)
Startup modelType of model: INSILICO MODEL / In silico model: Generated using Cryosparc
Initial angle assignmentType: RANDOM ASSIGNMENT
Software:
Namedetails
cryoSPARC (ver. 0.6.5)
RELION (ver. 2.1)
FREALIGN (ver. cisTEM 1.0)cisTEM 1.0
Final 3D classificationNumber classes: 1 / Avg.num./class: 196272 / Software - Name: FREALIGN (ver. cisTEM 1.0) / Software - details: cisTEM 1.0
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: FREALIGN (ver. cisTEM 1.0) / Software - details: cisTEM 1.0
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. cisTEM 1.0) / Software - details: cisTEM 1.0 / Number images used: 196272

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