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- PDB-6xqn: Structure of a mitochondrial calcium uniporter holocomplex (MICU1... -

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Basic information

Entry
Database: PDB / ID: 6xqn
TitleStructure of a mitochondrial calcium uniporter holocomplex (MICU1, MICU2, MCU, EMRE) in low Ca2+
Components
  • Calcium uniporter protein
  • Calcium uptake protein 1, mitochondrial
  • Calcium uptake protein 2, mitochondrial
  • Protein EMRE homolog, mitochondrial-like Protein
KeywordsTRANSPORT PROTEIN/CALCIUM BINDING PROTEIN / ion channel / calcium channel / mitochondrial calcium uniporter / MCU / EMRE / mitochondria / TRANSPORT PROTEIN-CALCIUM BINDING PROTEIN complex
Function / homology
Function and homology information


regulation of cellular hyperosmotic salinity response / negative regulation of mitochondrial calcium ion concentration / uniplex complex / calcium import into the mitochondrion / uniporter activity / positive regulation of mitochondrial calcium ion concentration / mitochondrial calcium ion transmembrane transport / mitochondrial calcium ion homeostasis / integral component of mitochondrial membrane / integral component of mitochondrial inner membrane ...regulation of cellular hyperosmotic salinity response / negative regulation of mitochondrial calcium ion concentration / uniplex complex / calcium import into the mitochondrion / uniporter activity / positive regulation of mitochondrial calcium ion concentration / mitochondrial calcium ion transmembrane transport / mitochondrial calcium ion homeostasis / integral component of mitochondrial membrane / integral component of mitochondrial inner membrane / calcium ion import / intracellular / calcium channel complex / calcium channel activity / protein homooligomerization / mitochondrial intermembrane space / defense response / mitochondrial inner membrane / protein heterodimerization activity / calcium ion binding / mitochondrion / identical protein binding
EF-hand domain / Calcium uptake protein 1/2/3 / MCU family / Essential MCU regulator, mitochondrial / EF-Hand 1, calcium-binding site / EF-hand domain pair / Calcium uniporter protein, C-terminal
Calcium uniporter protein / Protein EMRE homolog, mitochondrial-like Protein / Calcium uptake protein 2, mitochondrial / Calcium uptake protein 1, mitochondrial
Biological speciesTribolium castaneum (red flour beetle)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLong, S.B. / Wang, C. / Baradaran, R. / Jacewicz, A. / Delgado, B.
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: Elife / Year: 2020
Title: Structures reveal gatekeeping of the mitochondrial Ca uniporter by MICU1-MICU2.
Authors: Chongyuan Wang / Agata Jacewicz / Bryce D Delgado / Rozbeh Baradaran / Stephen Barstow Long /
Abstract: The mitochondrial calcium uniporter is a Ca-gated ion channel complex that controls mitochondrial Ca entry and regulates cell metabolism. MCU and EMRE form the channel while Ca-dependent regulation ...The mitochondrial calcium uniporter is a Ca-gated ion channel complex that controls mitochondrial Ca entry and regulates cell metabolism. MCU and EMRE form the channel while Ca-dependent regulation is conferred by MICU1 and MICU2 through an enigmatic process. We present a cryo-EM structure of an MCU-EMRE-MICU1-MICU2 holocomplex comprising MCU and EMRE subunits from the beetle Tribolium castaneum in complex with a human MICU1-MICU2 heterodimer at 3.3 Å resolution. With analogy to how neuronal channels are blocked by protein toxins, a uniporter interaction domain on MICU1 binds to a channel receptor site comprising MCU and EMRE subunits to inhibit ion flow under resting Ca conditions. A Ca-bound structure of MICU1-MICU2 at 3.1 Å resolution indicates how Ca-dependent changes enable dynamic response to cytosolic Ca signals.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJul 9, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 5, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Assembly

Deposited unit
E: Protein EMRE homolog, mitochondrial-like Protein
A: Calcium uniporter protein
B: Calcium uniporter protein
G: Protein EMRE homolog, mitochondrial-like Protein
C: Calcium uniporter protein
H: Protein EMRE homolog, mitochondrial-like Protein
D: Calcium uniporter protein
I: Calcium uptake protein 1, mitochondrial
J: Calcium uptake protein 2, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)207,09710
Polymers207,0569
Non-polymers401
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18620 Å2
ΔGint-194 kcal/mol
Surface area69930 Å2

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Components

#1: Protein Protein EMRE homolog, mitochondrial-like Protein / EMRE


Mass: 7372.190 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tribolium castaneum (red flour beetle) / Gene: TcasGA2_TC012057 / Production host: Homo sapiens (human) / References: UniProt: D6X268
#2: Protein
Calcium uniporter protein / MCU


Mass: 23634.100 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tribolium castaneum (red flour beetle) / Gene: TcasGA2_TC013837 / Production host: Homo sapiens (human) / References: UniProt: D6WIX5
#3: Protein Calcium uptake protein 1, mitochondrial / Atopy-related autoantigen CALC / ara CALC / Calcium-binding atopy-related autoantigen 1 / MICU1


Mass: 45422.883 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MICU1, CALC, CBARA1 / Production host: Homo sapiens (human) / References: UniProt: Q9BPX6
#4: Protein Calcium uptake protein 2, mitochondrial / EF-hand domain-containing family member A1 / MICU2


Mass: 44980.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MICU2, EFHA1 / Production host: Homo sapiens (human) / References: UniProt: Q8IYU8
#5: Chemical ChemComp-CA / CALCIUM ION / Calcium


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Mitochondrial Calcium Uniporter holocomplexCOMPLEX1, 2, 3, 40MULTIPLE SOURCES
2EMRECOMPLEX11RECOMBINANT
3MCUCOMPLEX21RECOMBINANT
4MICU1-MICU2 heterodimerCOMPLEX3, 41RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Tribolium castaneum (red flour beetle)7070
23Tribolium castaneum (red flour beetle)7070
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Homo sapiens (human)9606Expi293, Invitrogen
23Homo sapiens (human)9606Expi293, Invitrogen
34Homo sapiens (human)9606Expi293, Invitrogen
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2150 mMsodium chlorideNaClSodium chloride1
35 mMEGTA1
SpecimenConc.: 1 mg/ml / Details: Monodisperse sample / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 71 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 5 / Num. of real images: 21115
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
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
14PHENIX1.17.1-3660model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 17440131
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 350160 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 124 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient

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