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- PDB-6dnf: Cryo-EM structure of the mitochondrial calcium uniporter MCU from... -

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Entry
Database: PDB / ID: 6dnf
TitleCryo-EM structure of the mitochondrial calcium uniporter MCU from the fungus Cyphellophora europaea
ComponentsMitochondrial calcium uniporter MCU
KeywordsMEMBRANE PROTEIN / mitochondria / calcium / ion channel / eukaryotic
Function / homologyCalcium uniporter protein, C-terminal / MCU family / Mitochondrial calcium uniporter / mitochondrial calcium ion homeostasis / mitochondrion / integral component of membrane / identical protein binding / Uncharacterized protein
Function and homology information
Specimen sourceCyphellophora europaea CBS 101466 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.2 Å resolution
AuthorsLong, S.B. / 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
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jun 6, 2018 / Release: Jul 11, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jul 11, 2018Structure modelrepositoryInitial release
1.1Jul 25, 2018Structure modelData collection / Database referencescitation_citation.pdbx_database_id_PubMed / _citation.title
1.2Aug 8, 2018Structure modelData collection / Database referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)164,54915
Polyers158,5494
Non-polymers6,00011
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide
Mitochondrial calcium uniporter MCU


Mass: 39637.176 Da / Num. of mol.: 4
Source: (gene. exp.) Cyphellophora europaea CBS 101466 (fungus)
Gene: HMPREF1541_00236 / Production host: Komagataella pastoris (fungus) / References: UniProt: W2SDE2
#2: Chemical
ChemComp-DGG / 1-[GLYCEROLYLPHOSPHONYL]-2-[8-(2-HEXYL-CYCLOPROPYL)-OCTANAL-1-YL]-3-[HEXADECANAL-1-YL]-GLYCEROL


Mass: 734.981 Da / Num. of mol.: 8 / Formula: C39H75O10P
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / 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 (MCU) from the fungus Cyphellophora europaea.
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.158328 MDa / Experimental value: NO
Source (natural)Organism: Cyphellophora europaea (fungus)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHepes1
2150 mMsodium chlorideNaCl1
31 mMcalcium chlorideCaCl21
40.5 mMdigitonindigitonin1
50.05 mg/mL1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerolcardiolipin1
SpecimenConc.: 5 / Details: Monodisperse sample / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 / Details: 2 second blot, blot force of 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.2 / Electron dose: 2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 3 / Number of real images: 6040
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategoryDetails
1RELION2.1particle selection
2SerialEMimage acquisition
3RELION2.1image acquisition
4EMAN2image acquisition
5FREALIGNimage acquisitioncisTEM 1.0
7CTFFIND4.1.5CTF correction
10Coot0.8.8model fitting
12PHENIX1.13-2998model refinement
13RELION2.1initial Euler assignment
14FREALIGNinitial Euler assignmentcisTEM 1.0
15FREALIGN1.0final Euler assignmentcisTEM 1.0
16FREALIGN1.0classificationcisTEM 1.0
17FREALIGN3D reconstructioncisTEM 1.0
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 547637
SymmetryPoint symmetry: C2
3D reconstructionResolution: 3.2 / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 376541 / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingOverall b value: 136.58 / Ref protocol: AB INITIO MODEL / Ref space: REAL / Target criteria: Correlation coefficient

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