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- PDB-6x4s: MCU-EMRE complex of a metazoan mitochondrial calcium uniporter -

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Basic information

Entry
Database: PDB / ID: 6x4s
TitleMCU-EMRE complex of a metazoan mitochondrial calcium uniporter
ComponentsCalcium uniporter protein,Protein EMRE homolog, mitochondrial-like Protein fusion
KeywordsMEMBRANE PROTEIN / ion channel / calcium channel / mitochondrial calcium uniporter / MCU / EMRE / mitochondria
Function / homology
Function and homology information


uniporter activity / uniplex complex / calcium import into the mitochondrion / mitochondrial calcium ion homeostasis / calcium channel activity
Similarity search - Function
Essential MCU regulator, mitochondrial / Putative mitochondrial precursor protein / Calcium uniporter protein, C-terminal / MCU family / Mitochondrial calcium uniporter
Similarity search - Domain/homology
Calcium uniporter protein / Essential MCU regulator, mitochondrial
Similarity search - Component
Biological speciesTribolium castaneum (red flour beetle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLong, S.B. / Wang, C. / Baradaran, R.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131921 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P30CA008748 United States
CitationJournal: J Mol Biol / Year: 2020
Title: Structure and Reconstitution of an MCU-EMRE Mitochondrial Ca Uniporter Complex.
Authors: Chongyuan Wang / Rozbeh Baradaran / Stephen Barstow Long /
Abstract: The proteins MCU and EMRE form the minimal functional unit of the mitochondrial calcium uniporter complex in metazoans, a highly selective and tightly controlled Ca channel of the inner mitochondrial ...The proteins MCU and EMRE form the minimal functional unit of the mitochondrial calcium uniporter complex in metazoans, a highly selective and tightly controlled Ca channel of the inner mitochondrial membrane that regulates cellular metabolism. Here we present functional reconstitution of an MCU-EMRE complex from the red flour beetle, Tribolium castaneum, and a cryo-EM structure of the complex at 3.5 Å resolution. Using a novel assay, we demonstrate robust Ca uptake into proteoliposomes containing the purified complex. Uptake is dependent on EMRE and also on the mitochondrial lipid cardiolipin. The structure reveals a tetrameric channel with a single ion pore. EMRE is located at the periphery of the transmembrane domain and associates primarily with the first transmembrane helix of MCU. Coiled-coil and juxtamembrane domains within the matrix portion of the complex adopt markedly different conformations than in a structure of a human MCU-EMRE complex, suggesting that the structures represent different conformations of these functionally similar metazoan channels.
History
DepositionMay 22, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 2, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 30, 2020Group: Database references / Category: citation / Item: _citation.title
Revision 1.2Oct 14, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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  • EMDB-22042
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Assembly

Deposited unit
A: Calcium uniporter protein,Protein EMRE homolog, mitochondrial-like Protein fusion
B: Calcium uniporter protein,Protein EMRE homolog, mitochondrial-like Protein fusion
C: Calcium uniporter protein,Protein EMRE homolog, mitochondrial-like Protein fusion
D: Calcium uniporter protein,Protein EMRE homolog, mitochondrial-like Protein fusion
E: Calcium uniporter protein,Protein EMRE homolog, mitochondrial-like Protein fusion
F: Calcium uniporter protein,Protein EMRE homolog, mitochondrial-like Protein fusion
G: Calcium uniporter protein,Protein EMRE homolog, mitochondrial-like Protein fusion
H: Calcium uniporter protein,Protein EMRE homolog, mitochondrial-like Protein fusion
hetero molecules


Theoretical massNumber of molelcules
Total (without water)220,2869
Polymers220,2468
Non-polymers401
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17060 Å2
ΔGint-178 kcal/mol
Surface area43130 Å2

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Components

#1: Protein
Calcium uniporter protein,Protein EMRE homolog, mitochondrial-like Protein fusion


Mass: 27530.723 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tribolium castaneum (red flour beetle) / Gene: TcasGA2_TC013837, TcasGA2_TC012057 / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: D6WIX5, UniProt: D6X268
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Mitochondrial Calcium Uniporter (MCU) in complex with EMRE. Embedded in lipid nanodiscs containing cardiolipin.
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Tribolium castaneum (red flour beetle)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293, Invitrogen
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHepes1
2150 mMsodium chlorideNaClSodium chloride1
31 mMcalcium chlorideCaCl21
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: 2 second blot, blot force of 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Nominal defocus max: -3000 nm / Nominal defocus min: -1000 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.25 sec. / Electron dose: 1.9 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 8143
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
2SerialEMimage acquisition
4CTFFIND4.1.5CTF correction
7Coot0.8.8model fitting
9cryoSPARCv2.12.4initial Euler assignment
10RELION3initial Euler assignment
11RELION3final Euler assignment
12cryoSPARCv2.12.4classification
13RELION33D reconstruction
20PHENIX1.17.1-3660model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4460801
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52493 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 127 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0075496
ELECTRON MICROSCOPYf_angle_d0.837501
ELECTRON MICROSCOPYf_dihedral_angle_d13.795750
ELECTRON MICROSCOPYf_chiral_restr0.05927
ELECTRON MICROSCOPYf_plane_restr0.005914

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