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- PDB-6xpd: Cryo-EM structure of human ZnT8 double mutant - D110N and D224N, ... -

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Basic information

Entry
Database: PDB / ID: 6xpd
TitleCryo-EM structure of human ZnT8 double mutant - D110N and D224N, determined in outward-facing conformation
ComponentsZinc transporter 8
KeywordsTRANSPORT PROTEIN / ZnT8 / zinc transporter
Function / homology
Function and homology information


zinc ion import across plasma membrane / Zinc efflux and compartmentalization by the SLC30 family / insulin processing / zinc ion import into organelle / zinc:proton antiporter activity / zinc ion transmembrane transporter activity / zinc ion transport / zinc ion transmembrane transport / regulation of vesicle-mediated transport / intracellular zinc ion homeostasis ...zinc ion import across plasma membrane / Zinc efflux and compartmentalization by the SLC30 family / insulin processing / zinc ion import into organelle / zinc:proton antiporter activity / zinc ion transmembrane transporter activity / zinc ion transport / zinc ion transmembrane transport / regulation of vesicle-mediated transport / intracellular zinc ion homeostasis / insulin secretion / response to zinc ion / transport vesicle membrane / Insulin processing / response to type II interferon / response to glucose / response to interleukin-1 / secretory granule membrane / secretory granule / positive regulation of insulin secretion / cytoplasmic vesicle / Golgi membrane / intracellular membrane-bounded organelle / protein homodimerization activity / zinc ion binding / plasma membrane
Similarity search - Function
: / Cation efflux protein, cytoplasmic domain superfamily / Cation efflux protein / Cation efflux transmembrane domain superfamily / Cation efflux family
Similarity search - Domain/homology
Proton-coupled zinc antiporter SLC30A8
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsBai, X.C. / Xue, J. / Jiang, Y.X.
CitationJournal: Elife / Year: 2020
Title: Cryo-EM structures of human ZnT8 in both outward- and inward-facing conformations.
Authors: Jing Xue / Tian Xie / Weizhong Zeng / Youxing Jiang / Xiao-Chen Bai /
Abstract: ZnT8 is a Zn/H antiporter that belongs to SLC30 family and plays an essential role in regulating Zn accumulation in the insulin secretory granules of pancreatic β cells. However, the Zn/H exchange ...ZnT8 is a Zn/H antiporter that belongs to SLC30 family and plays an essential role in regulating Zn accumulation in the insulin secretory granules of pancreatic β cells. However, the Zn/H exchange mechanism of ZnT8 remains unclear due to the lack of high-resolution structures. Here, we report the cryo-EM structures of human ZnT8 (HsZnT8) in both outward- and inward-facing conformations. HsZnT8 forms a dimeric structure with four Zn binding sites within each subunit: a highly conserved primary site in transmembrane domain (TMD) housing the Zn substrate; an interfacial site between TMD and C-terminal domain (CTD) that modulates the Zn transport activity of HsZnT8; and two adjacent sites buried in the cytosolic domain and chelated by conserved residues from CTD and the His-Cys-His (HCH) motif from the N-terminal segment of the neighboring subunit. A comparison of the outward- and inward-facing structures reveals that the TMD of each HsZnT8 subunit undergoes a large structural rearrangement, allowing for alternating access to the primary Zn site during the transport cycle. Collectively, our studies provide the structural insights into the Zn/H exchange mechanism of HsZnT8.
History
DepositionJul 8, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 5, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 12, 2020Group: 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.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)70,4346
Polymers70,1722
Non-polymers2624
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5890 Å2
ΔGint-192 kcal/mol
Surface area29600 Å2

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Components

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


Mass: 35086.160 Da / Num. of mol.: 2 / Mutation: D110N, D224N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line: HEK293 / Gene: SLC30A8, ZNT8 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q8IWU4
#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: Human Znt8 double mutant - D110N and D224N / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.035 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human) / Cellular location: Insulin secretory granule
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 7.4
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 FIELD / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV / Phase plate: VOLTA PHASE PLATE

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
7Cootmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55851 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0074734
ELECTRON MICROSCOPYf_angle_d0.7016444
ELECTRON MICROSCOPYf_dihedral_angle_d15.308632
ELECTRON MICROSCOPYf_chiral_restr0.041802
ELECTRON MICROSCOPYf_plane_restr0.003778

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