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- EMDB-33619: Cryo-EM structure of a eukaryotic ZnT8 in the presence of zinc -

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Basic information

Entry
Database: EMDB / ID: EMD-33619
TitleCryo-EM structure of a eukaryotic ZnT8 in the presence of zinc
Map dataxtZnT8-Zn2 in the presence of 1 mM Zn2
Sample
  • Complex: xtZnT8 dimer complexed with zinc
    • Protein or peptide: Zinc transporter 8
  • Ligand: ZINC ION
KeywordsZinc transporter / SLC30 / ZnT / CDF / Proton-coupled antiporter / MEMBRANE PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.85 Å
AuthorsZhang S / Fu C / Luo Y / Sun Z / Su Z / Zhou X
Funding support China, 1 items
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, 2022-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33619.png

Downloads & links

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Map

FileDownload / File: emd_33619.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationxtZnT8-Zn2 in the presence of 1 mM Zn2
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 192 pix.
= 211.2 Å		1.1 Å/pix.
x 192 pix.
= 211.2 Å		1.1 Å/pix.
x 192 pix.
= 211.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-4.6573453 - 7.3341174
Average (Standard dev.)0.12475715 (±0.20173211)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 211.20001 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_33619_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33619_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : xtZnT8 dimer complexed with zinc

EntireName: xtZnT8 dimer complexed with zinc
Components
  • Complex: xtZnT8 dimer complexed with zinc
    • Protein or peptide: Zinc transporter 8
  • Ligand: ZINC ION

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Supramolecule #1: xtZnT8 dimer complexed with zinc

SupramoleculeName: xtZnT8 dimer complexed with zinc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog)

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Macromolecule #1: Zinc transporter 8

MacromoleculeName: Zinc transporter 8 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog)
Molecular weightTheoretical: 41.539031 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MKGSEEAYLV SDKATKMYSL TKDSEKNHPS KPPLQDEENP QSKYHCHNNN KKAYDARQRE QTFAKKKLCI ASLICFVFIS AEIVGGYIA GSLAVVTDAA HLLVDLSSFF ISLCSLWLSS KSSTTRLTFG WHRAEILGAL MSVITIWLVT GVLVYLACER L IRPDYTID ...String:
MKGSEEAYLV SDKATKMYSL TKDSEKNHPS KPPLQDEENP QSKYHCHNNN KKAYDARQRE QTFAKKKLCI ASLICFVFIS AEIVGGYIA GSLAVVTDAA HLLVDLSSFF ISLCSLWLSS KSSTTRLTFG WHRAEILGAL MSVITIWLVT GVLVYLACER L IRPDYTID GTVMLITSAC ALGANLVLAL ILHQSGHGHS HAGGKHEHMA SEYKPQTNAS IRAAFIHVIG DLFQSISVLI SA LIIYFKP EYKMADPICT FIFSIFVLIT TVTVLRDLLT VLMEGTPRGI HYSDVKQSIL AVDGVKSVHS LHLWALTMNQ VIL SAHIAT DIVGESKRIL KDVTQNVFAR FPFHSVTIQV EPIEDQSPEC MFCYEPTQ

UniProtKB: Proton-coupled zinc antiporter SLC30A8

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: ZN
Molecular weightTheoretical: 65.409 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

Concentration8.0 mg/mL
BufferpH: 7.5
Details: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM 2-mercaptoethanol, 0.5 mM DDM and 1 mM ZnSO4
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 2-3 s before plunging.
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: -1.7 µm / Nominal defocus min: -1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 928822
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6xpe

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 233408
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6xpe


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-7y5g:
Cryo-EM structure of a eukaryotic ZnT8 in the presence of zinc

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