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- EMDB-13987: Cryo-EM map of magnesium-bound EleNRMT in complex with two nanobo... -

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Basic information

Entry
Database: EMDB / ID: EMD-13987
TitleCryo-EM map of magnesium-bound EleNRMT in complex with two nanobodies at 4.1A
Map data
Sample
  • Complex: Magnesium-bound EleNRMT in complex with two nanobodies
    • Organelle or cellular component: Magnesium-bound EleNRMT
      • Protein or peptide: Divalent metal cation transporter
    • Organelle or cellular component: Nanobody 1Single-domain antibody
      • Protein or peptide: Nanobody 1Single-domain antibody
    • Organelle or cellular component: Nanobody 2Single-domain antibody
      • Protein or peptide: Nanobody 2Single-domain antibody
  • Ligand: MAGNESIUM ION
Function / homologyNRAMP family / Natural resistance-associated macrophage protein-like / metal ion transmembrane transporter activity / membrane / Divalent metal cation transporter
Function and homology information
Biological speciesEggerthella lenta (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsRamanadane K / Straub MS / Dutzler R / Manatschal C
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Elife / Year: 2022
Title: Structural and functional properties of a magnesium transporter of the SLC11/NRAMP family.
Authors: Karthik Ramanadane / Monique S Straub / Raimund Dutzler / Cristina Manatschal /
Abstract: Members of the ubiquitous SLC11/NRAMP family catalyze the uptake of divalent transition metal ions into cells. They have evolved to efficiently select these trace elements from a large pool of Ca and ...Members of the ubiquitous SLC11/NRAMP family catalyze the uptake of divalent transition metal ions into cells. They have evolved to efficiently select these trace elements from a large pool of Ca and Mg, which are both orders of magnitude more abundant, and to concentrate them in the cytoplasm aided by the cotransport of H serving as energy source. In the present study, we have characterized a member of a distant clade of the family found in prokaryotes, termed NRMTs, that were proposed to function as transporters of Mg. The protein transports Mg and Mn but not Ca by a mechanism that is not coupled to H. Structures determined by cryo-EM and X-ray crystallography revealed a generally similar protein architecture compared to classical NRAMPs, with a restructured ion binding site whose increased volume provides suitable interactions with ions that likely have retained much of their hydration shell.
History
DepositionDec 14, 2021-
Header (metadata) releaseDec 29, 2021-
Map releaseDec 29, 2021-
UpdateFeb 2, 2022-
Current statusFeb 2, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.263
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.263
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7qic
  • Surface level: 0.263
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13987.png

Downloads & links

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Map

FileDownload / File: emd_13987.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 180 pix.
= 234.36 Å		1.3 Å/pix.
x 180 pix.
= 234.36 Å		1.3 Å/pix.
x 180 pix.
= 234.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.302 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.263 / Movie #1: 0.263
Minimum - Maximum-1.2016681 - 1.5761361
Average (Standard dev.)0.00063417066 (±0.035505686)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 234.36002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3021.3021.302
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z234.360234.360234.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-1.2021.5760.001

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Supplemental data

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Mask #1

Fileemd_13987_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A

Fileemd_13987_half_map_1.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B

Fileemd_13987_half_map_2.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Magnesium-bound EleNRMT in complex with two nanobodies

EntireName: Magnesium-bound EleNRMT in complex with two nanobodies
Components
  • Complex: Magnesium-bound EleNRMT in complex with two nanobodies
    • Organelle or cellular component: Magnesium-bound EleNRMT
      • Protein or peptide: Divalent metal cation transporter
    • Organelle or cellular component: Nanobody 1Single-domain antibody
      • Protein or peptide: Nanobody 1Single-domain antibody
    • Organelle or cellular component: Nanobody 2Single-domain antibody
      • Protein or peptide: Nanobody 2Single-domain antibody
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Magnesium-bound EleNRMT in complex with two nanobodies

SupramoleculeName: Magnesium-bound EleNRMT in complex with two nanobodies
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 0.013 kDa/nm

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Supramolecule #2: Magnesium-bound EleNRMT

SupramoleculeName: Magnesium-bound EleNRMT / type: organelle_or_cellular_component / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: thermostabilized version
Source (natural)Organism: Eggerthella lenta (bacteria)
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)

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Supramolecule #3: Nanobody 1

SupramoleculeName: Nanobody 1 / type: organelle_or_cellular_component / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)

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Supramolecule #4: Nanobody 2

SupramoleculeName: Nanobody 2 / type: organelle_or_cellular_component / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)

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Macromolecule #1: Divalent metal cation transporter

MacromoleculeName: Divalent metal cation transporter / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Eggerthella lenta (bacteria)
Molecular weightTheoretical: 46.848828 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: MMMKKNEKLE LRDVAVDAAE STELVEVPEK KQPKKIWLLL AALGPGIVTA MAGNDAGGIS TYSTVGAKFG FATLWVIPIM CVLLIVVQM TAARMGAVTG KGFAALIRER FGIRLTALAM LALLIGNVAT TFSEFAGIAS GMEMFGVSKY LSVPVAAVAV W LLVVGGSY ...String:
MMMKKNEKLE LRDVAVDAAE STELVEVPEK KQPKKIWLLL AALGPGIVTA MAGNDAGGIS TYSTVGAKFG FATLWVIPIM CVLLIVVQM TAARMGAVTG KGFAALIRER FGIRLTALAM LALLIGNVAT TFSEFAGIAS GMEMFGVSKY LSVPVAAVAV W LLVVGGSY KRVEKVFLIL SLVFVTYIVA AFMAQPNWEE ALTSTVVPHI VNDQSFVSLV IAMIGTTIAP WMMFFNQSNV VE KGVTVKD LFSQKVDVVA GTIAACLVAW FIIVTTGAVL FPQGIEIESA ADAARALAPF AGHYAEALFA IGLIAASFLA ACV LPLTTA FVICEAFGWE AGVSFKWKEA PLFKSIFTFV IAFSAVVVLI PNIDLMGVML TAQFVNGLIL PVLLVFMAII AADK RVMGA YRSRIVSRVL IWLTVGIVTV LTAALLVMQV LGI

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Macromolecule #2: Nanobody 1

MacromoleculeName: Nanobody 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.12676 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString:
QLQLVESGGG LVQPGGSLRL SCEASGKVFM INAMGWYRQA PGKQRELVAF ISRRGNINYA DSVKGRFTIS RDNAKNTVYL QMNSLRPED TAIYYCSADP RSNLDDGRYW GKGTPVTVSS

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Macromolecule #3: Nanobody 2

MacromoleculeName: Nanobody 2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.767339 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString:
QLQLVESGGG LVLAGGSLRL SCAASVRTFS HYALGWFRQA PGKEREFVAA IRWTGSSANY ADSVKGRFTI SRDNAKNTVD LRMNSLKPE DTAVYYCAAR TVYRPGFEDP NEYAYWGQGT RVTVSS

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration3.5 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chloridesodium chloride
20.0 mMHEPESHEPES
0.25 %DMDecylmaltopyranoside
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
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: 3 / Number real images: 12427 / Average exposure time: 1.01 sec. / Average electron dose: 69.554 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2582066
CTF correctionSoftware - Name: cryoSPARC (ver. 3.2)
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 100176
FSC plot (resolution estimation)

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