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- EMDB-41629: Cryo-EM structure of the human MRS2 magnesium channel under Mg2+ ... -

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Basic information

Entry
Database: EMDB / ID: EMD-41629
TitleCryo-EM structure of the human MRS2 magnesium channel under Mg2+ condition (C1 map)
Map datalocal resolution filtered map
Sample
  • Complex: Cryo-EM structure of the pentameric human MRS2 magnesium channel under Mg2+ condition at an average resolution of 2.8 A, filtered to local resolution, C1
    • Protein or peptide: Mitochondrial RNA Splicing 2
KeywordsMagnesium / Ion channel / Membrane protein / METAL TRANSPORT / Ion Translocation / Divalent Ion / Mg2+ / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLai LTF / Balaraman J / Zhou F / Matthies D
Funding support United States, 1 items
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: Lai LTF / Balaraman J / Zhou F / Matthies D
History
DepositionAug 16, 2023-
Header (metadata) releaseSep 13, 2023-
Map releaseSep 13, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41629.png

Downloads & links

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Map

FileDownload / File: emd_41629.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocal resolution filtered map
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 1.1
Minimum - Maximum-8.020273 - 9.972338000000001
Average (Standard dev.)0.005209426 (±0.15716347)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41629_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: unsharpened map

Fileemd_41629_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

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Density Histograms

Histogram

Histogram (log scale)

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Additional map: sharpened map

Fileemd_41629_additional_2.map
Annotationsharpened map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_41629_half_map_1.map
Annotationhalf map B
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_41629_half_map_2.map
Annotationhalf map A
Projections & Slices
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Sample components

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

EntireName: Cryo-EM structure of the pentameric human MRS2 magnesium channel under Mg2+ condition at an average resolution of 2.8 A, filtered to local resolution, C1
Components
  • Complex: Cryo-EM structure of the pentameric human MRS2 magnesium channel under Mg2+ condition at an average resolution of 2.8 A, filtered to local resolution, C1
    • Protein or peptide: Mitochondrial RNA Splicing 2

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

SupramoleculeName: Cryo-EM structure of the pentameric human MRS2 magnesium channel under Mg2+ condition at an average resolution of 2.8 A, filtered to local resolution, C1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 219 KDa

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Macromolecule #1: Mitochondrial RNA Splicing 2

MacromoleculeName: Mitochondrial RNA Splicing 2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MECLRSLPCL LPRAMRLPRR TLCALALDVT SVGPPVAACG RRANLIGRSR AAQLCGPDRL RVAGEVHRFR TSDVSQATLA SVAPVFTVT KFDKQGNVTS FERKKTELYQ ELGLQARDLR FQHVMSITVR NNRIIMRMEY LKAVITPECL LILDYRNLNL E QWLFRELP ...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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.3
Component:
ConcentrationName
20.0 mMHEPES
150.0 mMNaClSodium chloride
40.0 mMMgCl2
0.003 %LMNG

Details: 20 mM HEPES, 150 mM NaCl, 40 mM MgCl2, 0.003% LMNG, pH 7.3
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen 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 ...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..
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 105000
Specialist opticsEnergy 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 ...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, 3,991 and 9,656 movies were collected.
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm 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/Å2
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, 3,991 and 9,656 movies were collected.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1568444
Details: Good 2D class averages generated from ~1,000 manually picked particles served as templates for automatic particle picking.
Startup modelType of model: INSILICO MODEL
In silico model: 573,010 good particles selected from one round of 2D classification was subjected to ab-initio reconstruction (K=2) and heterogenous refinement (K=2) with C1 symmetry to further sort particles.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.3.2) / Details: SGD (stochastic gradient descent)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 3.3.2)
Details: 573,010 good particles selected from one round of 2D classification was subjected to ab-initio reconstruction (K=2) and heterogenous refinement (K=2) with C1 symmetry to further sort particles.
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.3.2)
Details: Non-uniform refinement was performed with 450,554 selected particles, followed by local motion correction and CTF refinement to correct for beam-tilt, spherical aberrations, and per-particle ...Details: Non-uniform refinement was performed with 450,554 selected particles, followed by local motion correction and CTF refinement to correct for beam-tilt, spherical aberrations, and per-particle defocus parameters. Non-uniform refinement with polished particles resulted in maps at 2.8 A (with C5 symmetry applied) and 3.1 A (with C1 symmetry applied), according to gold-standard FSC = 0.143 criterion.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2)
Details: Non-uniform refinement was performed with 450,554 selected particles, followed by local motion correction and CTF refinement to correct for beam-tilt, spherical aberrations, and per-particle ...Details: Non-uniform refinement was performed with 450,554 selected particles, followed by local motion correction and CTF refinement to correct for beam-tilt, spherical aberrations, and per-particle defocus parameters. Non-uniform refinement with polished particles resulted in maps at 2.8 A (with C5 symmetry applied) and 3.1 A (with C1 symmetry applied), according to gold-standard FSC = 0.143 criterion.
Number images used: 450554
DetailsCryo-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, 3,991 and 9,656 movies were collected.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3-f1


Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsThe model was then manually rebuilt in COOT v.0.9.7 using the local resolution filtered map, which was generated from refinement with C5 applied. The Mg2+ ions assigned in the pore regions were confirmed in the C1 map. Loop regions (residues 174-181, 273-287) were built with the unsharpened map. Iterative rounds of manual refinement in COOT and real-space refinement in Phenix v.1.20.1-4487 were performed.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 141

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