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

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Basic information

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 / homology
Function and homology information


uniporter activity / uniplex complex / mitochondrial calcium ion homeostasis / calcium import into the mitochondrion / calcium channel activity / protein homotetramerization / mitochondrial inner membrane / metal ion binding / identical protein binding
Similarity search - Function
Calcium uniporter protein, C-terminal / MCU family / Mitochondrial calcium uniporter
Similarity search - Domain/homology
Chem-DGG / Calcium uniporter protein, mitochondrial
Similarity search - Component
Biological speciesCyphellophora europaea CBS 101466 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLong, S.B. / Baradaran, R. / Wang, C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM094273 United States
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, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 11, 2018Provider: repository / Type: Initial release
Revision 1.1Jul 25, 2018Group: Data collection / Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Aug 8, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

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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
Polymers158,5494
Non-polymers6,00011
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Mitochondrial calcium uniporter MCU


Mass: 39637.176 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
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
Source method: isolated from a genetically manipulated source
Formula: C39H75O10P
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca

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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) 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 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 FIELD / Nominal magnification: 29000 X / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 2 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: 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
15FREALIGN1final Euler assignmentcisTEM 1.0
16FREALIGN1classificationcisTEM 1.0
17FREALIGN3D reconstructioncisTEM 1.0
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 547637
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 376541 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 136.58 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient

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