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- PDB-6d80: Cryo-EM structure of the mitochondrial calcium uniporter from N. ... -

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Basic information

Entry
Database: PDB / ID: 6d80
TitleCryo-EM structure of the mitochondrial calcium uniporter from N. fischeri bound to saposin
Components
  • Mitochondrial calcium uniporter
  • Saposin A
KeywordsTRANSPORT PROTEIN / Mitochondria / calcium channel
Function / homologyuniporter activity / Calcium uniporter protein, C-terminal / MCU family / Mitochondrial calcium uniporter / mitochondrial calcium ion homeostasis / calcium channel activity / mitochondrial inner membrane / Calcium uniporter protein
Function and homology information
Biological speciesHomo sapiens (human)
Aspergillus fischeri (mold)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5 Å
AuthorsNguyen, N.X. / Armache, J.-P. / Cheng, Y. / Bai, X.C.
Funding support United States, 3items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM079179 United States
Welch FoundationI-1578 United States
CitationJournal: Nature / Year: 2018
Title: Cryo-EM structure of a fungal mitochondrial calcium uniporter.
Authors: Nam X Nguyen / Jean-Paul Armache / Changkeun Lee / Yi Yang / Weizhong Zeng / Vamsi K Mootha / Yifan Cheng / Xiao-Chen Bai / Youxing Jiang /
Abstract: The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel localized to the inner mitochondrial membrane. Here, we describe the structure of an MCU orthologue from the fungus ...The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel localized to the inner mitochondrial membrane. Here, we describe the structure of an MCU orthologue from the fungus Neosartorya fischeri (NfMCU) determined to 3.8 Å resolution by phase-plate cryo-electron microscopy. The channel is a homotetramer with two-fold symmetry in its amino-terminal domain (NTD) that adopts a similar structure to that of human MCU. The NTD assembles as a dimer of dimers to form a tetrameric ring that connects to the transmembrane domain through an elongated coiled-coil domain. The ion-conducting pore domain maintains four-fold symmetry, with the selectivity filter positioned at the start of the pore-forming TM2 helix. The aspartate and glutamate sidechains of the conserved DIME motif are oriented towards the central axis and separated by one helical turn. The structure of NfMCU offers insights into channel assembly, selective calcium permeation, and inhibitor binding.
History
DepositionApr 25, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 11, 2018Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Data collection / Data processing / Category: em_3d_reconstruction / Item: _em_3d_reconstruction.resolution
Revision 1.2Jul 25, 2018Group: Data collection / Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Aug 1, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Feb 1, 2023Group: Database references / Structure summary / Category: audit_author / citation_author / database_2
Item: _audit_author.name / _citation_author.name ..._audit_author.name / _citation_author.name / _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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Assembly

Deposited unit
F: Saposin A
H: Saposin A
G: Saposin A
I: Saposin A
J: Saposin A
K: Saposin A
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)233,22111
Polymers233,18110
Non-polymers401
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: Assembly of the saposin A molecules was determined based on clear electron density in the cryo-EM map, low pass-filtered to 5 Angstrom resolution using the crystal structure of saposin A ...Evidence: Assembly of the saposin A molecules was determined based on clear electron density in the cryo-EM map, low pass-filtered to 5 Angstrom resolution using the crystal structure of saposin A (PDB ID: 2DOB) as the starting model for model building and refinement.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13970 Å2
ΔGint-91 kcal/mol
Surface area95800 Å2

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Components

#1: Protein
Saposin A


Mass: 6911.511 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein
Mitochondrial calcium uniporter /


Mass: 47928.004 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aspergillus fischeri (mold) / Gene: NFIA_105760 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A1CWT6
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / 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

Component
IDNameTypeEntity IDParent-IDSource
1Mitochondrial calcium uniporter / saposin complexORGANELLE OR CELLULAR COMPONENT#1-#20MULTIPLE SOURCES
2saposinProsaposinCOMPLEX#11RECOMBINANT
3Mitochondrial calcium uniporterORGANELLE OR CELLULAR COMPONENT#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Aspergillus fischeri (mold)36630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli BL21(DE3) (bacteria)469008
23Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.5
Details: 20 mM HEPES, pH 7.5, 300 mM sodium chloride, 1 mM calcium chloride, 2% glycerol
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: N. fischeri MCU was reconstituted into lipid using E. coli total lipids and human saposin A as the membrane scaffolding protein.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsPhase plate: VOLTA PHASE PLATE

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM softwareName: RELION / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83343 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00711474
ELECTRON MICROSCOPYf_angle_d1.09515630
ELECTRON MICROSCOPYf_dihedral_angle_d10.8746880
ELECTRON MICROSCOPYf_chiral_restr0.0561834
ELECTRON MICROSCOPYf_plane_restr0.0072042

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