EMDB-41628: Cryo-EM structure of the human MRS2 magnesium channel under Mg2+-...

Basic information

Entry

Database: EMDB / ID: EMD-41628

• Title: Cryo-EM structure of the human MRS2 magnesium channel under Mg2+-free condition
• Map data: local resolution filtered map

Sample

• Complex: 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
  • Protein or peptide: Magnesium transporter MRS2 homolog, mitochondrial
• Ligand: MAGNESIUM ION
• Ligand: water

• Keywords: Magnesium / Ion channel / Membrane protein / METAL TRANSPORT / Ion Translocation / Divalent Ion / Pentamer

Function / homology

Function:

mitochondrial magnesium ion transmembrane transport / Miscellaneous transport and binding events / magnesium ion transmembrane transporter activity / lactate metabolic process / transmembrane transport / mitochondrial inner membrane / mitochondrion

Domain/homology:

Magnesium transporter MRS2-like / Magnesium transporter MRS2-like

Component:

Magnesium transporter MRS2 homolog, mitochondrial

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 3.3 Å
• Authors: Lai LTF / Balaraman J / Zhou F / Matthies D

Funding support

United States, 1 items

Organization	Grant number	Country
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)	ZIA HD008998	United States

• Citation: Journal: 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 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

Deposition	Aug 16, 2023	-
Header (metadata) release	Sep 13, 2023	-
Map release	Sep 13, 2023	-
Update	May 1, 2024	-
Current status	May 1, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_41628.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: local resolution filtered map
• Voxel size: X=Y=Z: 0.83 Å

Density

Contour Level	By AUTHOR: 0.14
Minimum - Maximum	-1.3844217 - 1.6362184
Average (Standard dev.)	0.0006703754 (±0.031491783)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 320 320 320 Spacing 320 320 320
Cell	A=B=C: 265.6 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_41628_msk_1.map

Additional map: unsharpened map

• File: emd_41628_additional_1.map
• Annotation: unsharpened map

Additional map: sharpened map

• File: emd_41628_additional_2.map
• Annotation: sharpened map

Half map: half map B

• File: emd_41628_half_map_1.map
• Annotation: half map B

Half map: half map A

• File: emd_41628_half_map_2.map
• Annotation: half map A

Sample components

Entire : Cryo-EM structure of the pentameric human MRS2 magnesium channel ...

• Entire: Name: 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

Components

• Complex: 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
  • Protein or peptide: Magnesium transporter MRS2 homolog, mitochondrial
• Ligand: MAGNESIUM ION
• Ligand: water

Supramolecule #1: Cryo-EM structure of the pentameric human MRS2 magnesium channel ...

• Supramolecule: Name: 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 / ID: 1 / Parent: 0 / Macromolecule list: #1
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 219 KDa

Macromolecule #1: Magnesium transporter MRS2 homolog, mitochondrial

• Macromolecule: Name: Magnesium transporter MRS2 homolog, mitochondrial / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 51.373516 KDa
• Recombinant expression: Organism: Homo sapiens (human)

Sequence

String:

MECLRSLPCL LPRAMRLPRR TLCALALDVT SVGPPVAACG RRANLIGRSR AAQLCGPDRL RVAGEVHRFR TSDVSQATLA SVAPVFTVT KFDKQGNVTS FERKKTELYQ ELGLQARDLR FQHVMSITVR NNRIIMRMEY LKAVITPECL LILDYRNLNL E QWLFRELP SQLSGEGQLV TYPLPFEFRA IEALLQYWIN TLQGKLSILQ PLILETLDAL VDPKHSSVDR SKLHILLQNG KS LSELETD IKIFKESILE ILDEEELLEE LCVSKWSDPQ VFEKSSAGID HAEEMELLLE NYYRLADDLS NAARELRVLI DDS QSIIFI NLDSHRNVMM RLNLQLTMGT FSLSLFGLMG VAFGMNLESS LEEDHRIFWL ITGIMFMGSG LIWRRLLSFL GRQL EAPLP PMMASLPKKT LLADRSMELK NSLRLDGLGS GRSILTNRDY KDDDDK

UniProtKB: Magnesium transporter MRS2 homolog, mitochondrial

Macromolecule #2: MAGNESIUM ION

• Macromolecule: Name: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 3 / Formula: MG
• Molecular weight: Theoretical: 24.305 Da

Macromolecule #3: water

• Macromolecule: Name: water / type: ligand / ID: 3 / Number of copies: 66 / Formula: HOH
• Molecular weight: Theoretical: 18.015 Da

Chemical component information

ChemComp-HOH:
WATER

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.5 mg/mL

Buffer

pH: 7.3
Component:

Concentration	Name
20.0 mM	HEPES
150.0 mM	NaCl
1.0 mM	EDTA
0.003 %	LMNG

Details: 20 mM HEPES, 150 mM NaCl, 1mM EDTA, 0.003% LMNG, pH 7.3

• Grid: Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA EM GP; 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..
• Details: This sample was monodisperse

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV; 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.
• Image recording: Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 3991 / Average exposure time: 3.462 sec. / Average electron dose: 50.0 e/Ų; 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.
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 105000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• 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.
• Particle selection: Number selected: 4802706 ; Details: Good 2D class averages generated from ~1,000 manually picked particles served as templates for automatic particle picking.
• Startup model: Type of model: INSILICO MODEL; In silico model: 4,802,706 particles were picked and extracted at 4x binned pixel size of 3.32 A and sorted by one round of 2D classification, ending up with 1,755,556 particles. Ab-initio reconstruction (K=3) was performed with the subset of particles after 2D classification.
• Final reconstruction: Number classes used: 1 / Applied symmetry - Point group: C₅ (5 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2); 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.; Number images used: 1744117
• Initial angle assignment: Type: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.3.2) / Details: SGD (stochastic gradient descent)
• Final angle assignment: Type: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.3.2); 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.
• Final 3D classification: Number classes: 2 / Software - Name: cryoSPARC (ver. 3.3.2); Details: Ab-initio reconstruction (K=3) was performed with the subset of particles after 2D classification, followed byand heterogenous refinement (K=3) using all particles with C1 symmetry was applied to remove junk particles. Particles from one class (2,237,316 particles ) were re-extracted at 1x binned pixel size of 0.83 A and subjected to non-uniform refinement followed by another round of heterogenous refinement (K=2) to further sort particles.

Atomic model buiding 1

Initial model

PDB ID:

8tul

Chain - Source name: PDB / Chain - Initial model type: experimental model

• Refinement: Space: REAL / Protocol: RIGID BODY FIT / Overall B value: 141

Output model

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

Atomic model buiding 2

Initial model

PDB ID:

8tul

Chain - Source name: PDB / Chain - Initial model type: experimental model

• Details: For 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.
• Refinement: Space: REAL / Protocol: FLEXIBLE FIT / Overall B value: 141

Output model

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