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- PDB-7qia: Structure of apo-EleNRMT in complex with two nanobodies at 3.5A -

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Basic information

Entry
Database: PDB / ID: 7qia
TitleStructure of apo-EleNRMT in complex with two nanobodies at 3.5A
Components
  • Divalent metal cation transporter
  • Nanobody 1Single-domain antibody
  • Nanobody 2Single-domain antibody
KeywordsMEMBRANE PROTEIN / SLC11 / Magnesium / LeuT fold
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsRamanadane, K. / Straub, M.S. / Dutzler, R. / Manatschal, C.
Funding support Switzerland, 1items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 29, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 2, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Divalent metal cation transporter
C: Nanobody 1
B: Nanobody 2


Theoretical massNumber of molelcules
Total (without water)73,7433
Polymers73,7433
Non-polymers00
Water543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Divalent metal cation transporter


Mass: 46848.828 Da / Num. of mol.: 1
Mutation: E88Q, A151S, E193Q, R207H, S244T, I256V, S275A, V366I, V385I, V418L, V429A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Eggerthella lenta (bacteria) / Gene: C1853_09580, C1871_08405 / Production host: Escherichia coli MC1061 (bacteria) / References: UniProt: A0A369N1S1
#2: Antibody Nanobody 1 / Single-domain antibody


Mass: 13126.760 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli MC1061 (bacteria)
#3: Antibody Nanobody 2 / Single-domain antibody


Mass: 13767.339 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli MC1061 (bacteria)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O

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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-IDSourceDetails
1EleNRMT in complex with two nanobodiesCOMPLEX#1-#30RECOMBINANT
2EleNRMTORGANELLE OR CELLULAR COMPONENT#11RECOMBINANTthermostabilized version
3Nanobody 1Single-domain antibodyORGANELLE OR CELLULAR COMPONENT#21RECOMBINANT
4Nanobody 2Single-domain antibodyORGANELLE OR CELLULAR COMPONENT#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.073 kDa/nmNO
210.047 kDa/nmNO
310.013 kDa/nmNO
410.013 kDa/nmNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Eggerthella lenta (bacteria)84112
33synthetic construct (others)32630
44synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli MC1061 (bacteria)1211845
33Escherichia coli MC1061 (bacteria)1211845
44Escherichia coli MC1061 (bacteria)1211845
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaClSodium chloride1
220 mMHEPESHEPES1
30.25 %DecylmaltopyranosideDM1
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.15 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 / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.01 sec. / Electron dose: 69.725 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 3 / Num. of real images: 22117

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
1cryoSPARC3.2particle selection
2EPU2.9image acquisition
4cryoSPARC3.2CTF correction
7Cootmodel fitting
9cryoSPARC3.2initial Euler assignment
10cryoSPARC3.2final Euler assignment
11cryoSPARC3.2classification
12cryoSPARC3.23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4139894
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 453950 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 20.04 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00284942
ELECTRON MICROSCOPYf_angle_d0.58596721
ELECTRON MICROSCOPYf_chiral_restr0.0428795
ELECTRON MICROSCOPYf_plane_restr0.0048837
ELECTRON MICROSCOPYf_dihedral_angle_d4.2216686

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