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- PDB-8ont: Structure of Setaria italica NRAT in complex with a nanobody -

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Basic information

Entry
Database: PDB / ID: 8ont
TitleStructure of Setaria italica NRAT in complex with a nanobody
Components
  • NRAMP related aluminium transporter
  • Nanobody1
KeywordsTRANSPORT PROTEIN / NRAT / NRAMP / SLC11 / Metal uptake / Aluminium transporter
Function / homology
Function and homology information


aluminum ion transmembrane transporter activity / aluminum cation transport / manganese ion transmembrane transporter activity / cadmium ion transmembrane transporter activity / response to aluminum ion / plasma membrane
Similarity search - Function
NRAMP family / Natural resistance-associated macrophage protein-like
Similarity search - Domain/homology
DIUNDECYL PHOSPHATIDYL CHOLINE / Uncharacterized protein
Similarity search - Component
Biological speciesSetaria italica (foxtail millet)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsRamanadane, K. / Liziczai, M. / Markovic, D. / Straub, M.S. / Rosalen, G.T. / Udovcic, A. / Dutzler, R. / Manatschal, C.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
Other government Switzerland
CitationJournal: Elife / Year: 2023
Title: Structural and functional properties of a plant NRAMP-related aluminum transporter.
Authors: Karthik Ramanadane / Márton Liziczai / Dragana Markovic / Monique S Straub / Gian T Rosalen / Anto Udovcic / Raimund Dutzler / Cristina Manatschal /
Abstract: The transport of transition metal ions by members of the SLC11/NRAMP family constitutes a ubiquitous mechanism for the uptake of Fe and Mn across all kingdoms of life. Despite the strong conservation ...The transport of transition metal ions by members of the SLC11/NRAMP family constitutes a ubiquitous mechanism for the uptake of Fe and Mn across all kingdoms of life. Despite the strong conservation of the family, two of its branches have evolved a distinct substrate preference with one mediating Mg uptake in prokaryotes and another the transport of Al into plant cells. Our previous work on the SLC11 transporter from revealed the basis for its Mg selectivity (Ramanadane et al., 2022). Here, we have addressed the structural and functional properties of a putative Al transporter from . We show that the protein transports diverse divalent metal ions and binds the trivalent ions Al and Ga, which are both presumable substrates. Its cryo-electron microscopy (cryo-EM) structure displays an occluded conformation that is closer to an inward- than an outward-facing state, with a binding site that is remodeled to accommodate the increased charge density of its transported substrate.
History
DepositionApr 3, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 12, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: NRAMP related aluminium transporter
B: Nanobody1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)73,8423
Polymers73,2192
Non-polymers6231
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2700 Å2
ΔGint-6 kcal/mol
Surface area24790 Å2

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Components

#1: Protein NRAMP related aluminium transporter


Mass: 60147.227 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Setaria italica (foxtail millet) / Gene: 101781512, SETIT_1G098100v2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: K3YRE7
#2: Antibody Nanobody1


Mass: 13071.546 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Cell (production host): MC1061 / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-PLC / DIUNDECYL PHOSPHATIDYL CHOLINE


Mass: 622.834 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C32H65NO8P / Comment: phospholipid*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: Complex between SiNRAT and a nanobody used as a fiducial marker for structure determination
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
Source (natural)Organism: Setaria italica (foxtail millet)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 7
SpecimenConc.: 2.7 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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 69.68 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 300000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0054410
ELECTRON MICROSCOPYf_angle_d0.5095996
ELECTRON MICROSCOPYf_dihedral_angle_d7.641619
ELECTRON MICROSCOPYf_chiral_restr0.037711
ELECTRON MICROSCOPYf_plane_restr0.006733

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