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- PDB-6dt0: Cryo-EM structure of a mitochondrial calcium uniporter -

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Basic information

Entry
Database: PDB / ID: 6dt0
TitleCryo-EM structure of a mitochondrial calcium uniporter
ComponentsMitochondrial calcium uniporter
KeywordsTRANSPORT PROTEIN / mitochondrial calcium uniporter / calcium-selective ion channel / calcium uptake / uniporter
Function / homologyCalcium uniporter protein, C-terminal / MCU family / Mitochondrial calcium uniporter / uniplex complex / calcium import into the mitochondrion / mitochondrial calcium ion homeostasis / uniporter activity / integral component of mitochondrial inner membrane / calcium channel activity / Uncharacterized protein
Function and homology information
Specimen sourceNeurospora crassa (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.7 Å resolution
AuthorsYoo, J. / Wu, M. / Yin, Y. / Herzik, M.A.J. / Lander, G.C. / Lee, S.-Y.
CitationJournal: Science / Year: 2018
Title: Cryo-EM structure of a mitochondrial calcium uniporter.
Authors: Jiho Yoo / Mengyu Wu / Ying Yin / Mark A Herzik / Gabriel C Lander / Seok-Yong Lee
Abstract: Calcium transport plays an important role in regulating mitochondrial physiology and pathophysiology. The mitochondrial calcium uniporter (MCU) is a calcium-selective ion channel that is the primary ...Calcium transport plays an important role in regulating mitochondrial physiology and pathophysiology. The mitochondrial calcium uniporter (MCU) is a calcium-selective ion channel that is the primary mediator for calcium uptake into the mitochondrial matrix. Here, we present the cryo-electron microscopy structure of the full-length MCU from to an overall resolution of ~3.7 angstroms. Our structure reveals a tetrameric architecture, with the soluble and transmembrane domains adopting different symmetric arrangements within the channel. The conserved W-D-Φ-Φ-E-P-V-T-Y sequence motif of MCU pore forms a selectivity filter comprising two acidic rings separated by one helical turn along the central axis of the channel pore. The structure combined with mutagenesis gives insight into the basis of calcium recognition.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jun 14, 2018 / Release: Jul 11, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jul 11, 2018Structure modelrepositoryInitial release
1.1Aug 15, 2018Structure modelData collection / Database referencescitation / citation_author_citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
A: Mitochondrial calcium uniporter
B: Mitochondrial calcium uniporter
C: Mitochondrial calcium uniporter
D: Mitochondrial calcium uniporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)211,8335
Polyers211,7924
Non-polymers401
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)9090
ΔGint (kcal/M)-80
Surface area (Å2)56340

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Components

#1: Protein/peptide
Mitochondrial calcium uniporter /


Mass: 52948.117 Da / Num. of mol.: 4 / Fragment: UNP residues 46-493 / Mutation: Y232A / Source: (gene. exp.) Neurospora crassa (fungus) / Gene: NCU08166 / Production host: Escherichia coli (E. coli) / References: UniProt: Q7S4I4
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Formula: Ca / Calcium

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Mitochondrial Calcium Uniporter / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Source (natural)Organism: Neurospora crassa (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 3.2 mg/ml
Details: Final reconstruction comprises ~80% of MCU in nanodisc (crosslinked using BS3) and ~20% MCU in amphipol.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 kelvins / Details: 4 second blot time

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 65 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansSampling size: 5 microns / Width: 3710 / Height: 3838

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Processing

EM software
IDNameVersionCategory
1RELION2.1particle selection
2Leginon3.2image acquisition
4CTFFIND4CTF correction
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
12RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: 1,791,114 particles of MCU in amphipol, 1,895,054 particles of crosslinked MCU in nanodisc
Number of particles selected: 3686168
SymmetryPoint symmetry: C2
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 36537 / Symmetry type: POINT
Atomic model buildingOverall b value: 100

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