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- PDB-8tup: Cryo-EM structure of the human MRS2 magnesium channel under Mg2+-... -

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Basic information

Entry
Database: PDB / ID: 8tup
TitleCryo-EM structure of the human MRS2 magnesium channel under Mg2+-free condition
ComponentsMagnesium transporter MRS2 homolog, mitochondrial
KeywordsMETAL TRANSPORT / Magnesium / Ion channel / Membrane protein / Ion Translocation / Divalent Ion / Pentamer
Function / homologymitochondrial magnesium ion transmembrane transport / Magnesium transporter MRS2-like / Miscellaneous transport and binding events / magnesium ion transmembrane transporter activity / lactate metabolic process / transmembrane transport / mitochondrial inner membrane / mitochondrion / Magnesium transporter MRS2 homolog, mitochondrial
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLai, L.T.F. / Balaraman, J. / Zhou, F. / Matthies, D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)ZIA HD008998 United States
CitationJournal: Nat Commun / Year: 2023
Title: Cryo-EM structures of human magnesium channel MRS2 reveal gating and regulatory mechanisms.
Authors: Louis Tung Faat Lai / Jayashree Balaraman / Fei Zhou / Doreen Matthies /
Abstract: Magnesium ions (Mg) play an essential role in cellular physiology. In mitochondria, protein and ATP synthesis and various metabolic pathways are directly regulated by Mg. MRS2, a magnesium channel ...Magnesium ions (Mg) play an essential role in cellular physiology. In mitochondria, protein and ATP synthesis and various metabolic pathways are directly regulated by Mg. MRS2, a magnesium channel located in the inner mitochondrial membrane, mediates the influx of Mg into the mitochondrial matrix and regulates Mg homeostasis. Knockdown of MRS2 in human cells leads to reduced uptake of Mg into mitochondria and disruption of the mitochondrial metabolism. Despite the importance of MRS2, the Mg translocation and regulation mechanisms of MRS2 are still unclear. Here, using cryo-EM we report the structures of human MRS2 in the presence and absence of Mg at 2.8 Å and 3.3 Å, respectively. From the homo-pentameric structures, we identify R332 and M336 as major gating residues, which are then tested using mutagenesis and two cellular divalent ion uptake assays. A network of hydrogen bonds is found connecting the gating residue R332 to the soluble domain, potentially regulating the gate. Two Mg-binding sites are identified in the MRS2 soluble domain, distinct from the two sites previously reported in CorA, a homolog of MRS2 in prokaryotes. Altogether, this study provides the molecular basis for understanding the Mg translocation and regulatory mechanisms of MRS2.
History
DepositionAug 16, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 13, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Database references / Refinement description / Category: citation / citation_author / em_3d_fitting_list
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_3d_fitting_list.initial_refinement_model_id
Revision 1.2May 1, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Magnesium transporter MRS2 homolog, mitochondrial
B: Magnesium transporter MRS2 homolog, mitochondrial
C: Magnesium transporter MRS2 homolog, mitochondrial
D: Magnesium transporter MRS2 homolog, mitochondrial
E: Magnesium transporter MRS2 homolog, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)256,9408
Polymers256,8685
Non-polymers733
Water1,18966
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration, Size-exclusion chromatography of purified MRS2 shows a elution volume corresponding to protein size around 400 kDa., native gel ...Evidence: electron microscopy, not applicable, gel filtration, Size-exclusion chromatography of purified MRS2 shows a elution volume corresponding to protein size around 400 kDa., native gel electrophoresis, Purified MRS2 showed a band at slightly above 38 kDa, which is smaller than the full-length size at 51 kDa, suggesting the N-terminal MTP has been cleaved in purified MRS2. Native PAGE of purified MRS2 shows a major band between the native marker at 242 kDa and 480 kDa indicating MRS2 assembles into an oligomer.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Magnesium transporter MRS2 homolog, mitochondrial


Mass: 51373.516 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MRS2 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q9HD23
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 66 / Source method: isolated from a natural source / Formula: H2O
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: Cryo-EM structure of the pentameric human MRS2 magnesium channel under Mg2+-free condition at an average resolution of 3.3 A, filtered to local resolution, C5
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.219 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F / Plasmid: pCMV
Buffer solutionpH: 7.3
Details: 20 mM HEPES, 150 mM NaCl, 1mM EDTA, 0.003% LMNG, pH 7.3
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2150 mMNaClSodium chloride1
31 mMEDTAEthylenediaminetetraacetic acid1
40.003 %LMNG1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K
Details: 400-mesh R1.2/1.3 Cu grids (Quantifoil) were made hydrophilic by glow discharging for 60 seconds with a current of 15 mA in a PELCO easiGlow system. The cryo grids were produced using a ...Details: 400-mesh R1.2/1.3 Cu grids (Quantifoil) were made hydrophilic by glow discharging for 60 seconds with a current of 15 mA in a PELCO easiGlow system. The cryo grids were produced using a Leica EM GP2 (Leica). The chamber was kept at 4 C and set to 95% humidity. 3 microliter sample at 0.5 mg/ml was applied to a glow-discharged holey grid, blotted for 6 s, and plunge frozen into liquid ethane and stored in liquid nitrogen.

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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: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.462 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3991
Details: Cryo-EM datasets were acquired with SerialEM using a Titan Krios (FEI, now ThermoFisher Scientific) operated at 300 keV and equipped with an energy filter and K3 camera (Gatan Inc.). Movies ...Details: Cryo-EM datasets were acquired with SerialEM using a Titan Krios (FEI, now ThermoFisher Scientific) operated at 300 keV and equipped with an energy filter and K3 camera (Gatan Inc.). Movies of 50 frames with a dose of 1 e-/A2 per frame (50 e-/A2 total dose) were recorded at a nominal magnification of 105,000x, corresponding to a physical pixel size of 0.83 A/px (super-resolution pixel size 0.415 A/px) in CDS mode at a dose rate of 10 e-/px/s and a defocus range of -0.7 to -2.0 um. In total, 9,656 movies were collected.
EM imaging opticsEnergyfilter name: GIF Bioquantum
Details: Cryo-EM datasets were acquired with SerialEM using a Titan Krios (FEI, now ThermoFisher Scientific) operated at 300 keV and equipped with an energy filter and K3 camera (Gatan Inc.). Movies ...Details: Cryo-EM datasets were acquired with SerialEM using a Titan Krios (FEI, now ThermoFisher Scientific) operated at 300 keV and equipped with an energy filter and K3 camera (Gatan Inc.). Movies of 50 frames with a dose of 1 e-/A2 per frame (50 e-/A2 total dose) were recorded at a nominal magnification of 105,000x, corresponding to a physical pixel size of 0.83 A/px (super-resolution pixel size 0.415 A/px) in CDS mode at a dose rate of 10 e-/px/s and a defocus range of -0.7 to -2.0 um. In total, 9,656 movies were collected.
Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryFitting-ID
1cryoSPARC3.3.2particle selection
3SerialEMimage acquisition
5cryoSPARC3.3.2CTF correction
8UCSF Chimera1.16model fitting1
13Cootmodel fitting2
14Cootmodel refinement2
15PHENIX1.20.1-4487model refinement2
16cryoSPARC3.3.2initial Euler assignment
18cryoSPARC3.3.2final Euler assignment
20cryoSPARC3.3.2classification
22cryoSPARC3.3.23D reconstruction
Image processingDetails: Cryo-EM datasets were acquired with SerialEM using a Titan Krios (FEI, now ThermoFisher Scientific) operated at 300 keV and equipped with an energy filter and K3 camera (Gatan Inc.). Movies ...Details: Cryo-EM datasets were acquired with SerialEM using a Titan Krios (FEI, now ThermoFisher Scientific) operated at 300 keV and equipped with an energy filter and K3 camera (Gatan Inc.). Movies of 50 frames with a dose of 1 e-/A2 per frame (50 e-/A2 total dose) were recorded at a nominal magnification of 105,000x, corresponding to a physical pixel size of 0.83 A/px (super-resolution pixel size 0.415 A/px) in CDS mode at a dose rate of 10 e-/px/s and a defocus range of -0.7 to -2.0 um. In total, 9,656 movies were collected.
CTF correctionDetails: Patch CTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4802706
Details: Good 2D class averages generated from ~1,000 manually picked particles served as templates for automatic particle picking.
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1744117 / Algorithm: BACK PROJECTION
Details: Final non-uniform refinement by using 1,744,117 particles yielded maps at 3.3 A (with C5 symmetry applied) and 3.6 A (with C1 symmetry applied) according to gold-standard FSC = 0.143 criterion.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
IDB valueProtocolSpaceDetails
1141RIGID BODY FITREAL
2141FLEXIBLE FITREALFor the MRS2-EDTA structures, models were generated from multiple rounds of manual refinement and real-space refinements with the final model of MRS2-Mg2+ as initial model.
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
18TUL

8tul

18TUL1PDBexperimental model
28TUL

8tul

28TUL1PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00213025
ELECTRON MICROSCOPYf_angle_d0.37217595
ELECTRON MICROSCOPYf_dihedral_angle_d3.0941710
ELECTRON MICROSCOPYf_chiral_restr0.0362025
ELECTRON MICROSCOPYf_plane_restr0.0032240

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