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- PDB-7y5h: Cryo-EM structure of a eukaryotic ZnT8 at a low pH -

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Basic information

Entry
Database: PDB / ID: 7y5h
TitleCryo-EM structure of a eukaryotic ZnT8 at a low pH
ComponentsZinc transporter 8
KeywordsMEMBRANE PROTEIN / Zinc transporter / SLC30 / ZnT / CDF / Proton-coupled antiporter
Function / homology
Function and homology information


Insulin processing / Zinc efflux and compartmentalization by the SLC30 family / zinc:proton antiporter activity / zinc ion import into organelle / transport vesicle membrane / secretory granule membrane / metal ion binding / plasma membrane
Similarity search - Function
: / Cation efflux protein, cytoplasmic domain superfamily / Cation efflux protein / Cation efflux transmembrane domain superfamily / Cation efflux transmembrane domain
Similarity search - Domain/homology
Proton-coupled zinc antiporter SLC30A8
Similarity search - Component
Biological speciesXenopus tropicalis (tropical clawed frog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.72 Å
AuthorsZhang, S. / Fu, C. / Luo, Y. / Sun, Z. / Su, Z. / Zhou, X.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31770783 China
CitationJournal: J Struct Biol / Year: 2023
Title: Cryo-EM structure of a eukaryotic zinc transporter at a low pH suggests its Zn-releasing mechanism.
Authors: Senfeng Zhang / Chunting Fu / Yongbo Luo / Qingrong Xie / Tong Xu / Ziyi Sun / Zhaoming Su / Xiaoming Zhou /
Abstract: Zinc transporter 8 (ZnT8) is mainly expressed in pancreatic islet β cells and is responsible for H-coupled uptake (antiport) of Zn into the lumen of insulin secretory granules. Structures of human ...Zinc transporter 8 (ZnT8) is mainly expressed in pancreatic islet β cells and is responsible for H-coupled uptake (antiport) of Zn into the lumen of insulin secretory granules. Structures of human ZnT8 and its prokaryotic homolog YiiP have provided structural basis for constructing a plausible transport cycle for Zn. However, the mechanistic role that protons play in the transport process remains unclear. Here we present a lumen-facing cryo-EM structure of ZnT8 from Xenopus tropicalis (xtZnT8) in the presence of Zn at a luminal pH (5.5). Compared to a Zn-bound xtZnT8 structure at a cytosolic pH (7.5), the low-pH structure displays an empty transmembrane Zn-binding site with a disrupted coordination geometry. Combined with a Zn-binding assay our data suggest that protons may disrupt Zn coordination at the transmembrane Zn-binding site in the lumen-facing state, thus facilitating Zn release from ZnT8 into the lumen.
History
DepositionJun 17, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 21, 2022Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2Jul 3, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Zinc transporter 8
A: Zinc transporter 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)83,3406
Polymers83,0782
Non-polymers2624
Water00
1


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

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Components

#1: Protein Zinc transporter 8 / ZnT-8 / Solute carrier family 30 member 8


Mass: 41539.031 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus tropicalis (tropical clawed frog)
Gene: slc30a8 / Production host: Komagataella pastoris (fungus) / References: UniProt: Q5XHB4
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: xtZnT8 dimer complexed with zinc and proton / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 5.5
Details: 20 mM MES-Na pH 5.5, 150 mM NaCl, 5 mM 2-mercaptoethanol and 0.5 mM DDM
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K / Details: blot for 2-3 s before plunging

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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 FIELD / Nominal defocus max: -1700 nm / Nominal defocus min: -1100 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 63.9 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPU2image acquisition
7Coot0.9.3model fitting
9PHENIX1.19.1model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13cryoSPARC3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 684578
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 101706 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6XPE

6xpe


Pdb chain-ID: A / Accession code: 6XPE / Source name: PDB / Type: experimental model

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