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- PDB-8f6j: Cryo-EM structure of a Zinc-loaded D287A mutant of the YiiP-Fab c... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8f6j | |||||||||
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Title | Cryo-EM structure of a Zinc-loaded D287A mutant of the YiiP-Fab complex | |||||||||
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![]() | TRANSPORT PROTEIN / Zinc transporter / cation diffusion facilitator / membrane protein | |||||||||
Function / homology | ![]() zinc efflux active transmembrane transporter activity / cadmium ion transmembrane transporter activity / ferrous iron transmembrane transporter activity / intracellular zinc ion homeostasis / metal ion binding / plasma membrane Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å | |||||||||
![]() | Lopez-Redondo, M.L. / Hussein, A.K. / Stokes, D.L. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Energy coupling and stoichiometry of Zn/H antiport by the prokaryotic cation diffusion facilitator YiiP. Authors: Adel Hussein / Shujie Fan / Maria Lopez-Redondo / Ian Kenney / Xihui Zhang / Oliver Beckstein / David L Stokes / ![]() Abstract: YiiP from Shewanella oneidensis is a prokaryotic Zn/H antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for ...YiiP from Shewanella oneidensis is a prokaryotic Zn/H antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for homeostasis of transition metal ions. Previous studies of YiiP as well as related CDF transporters have established a homodimeric architecture and the presence of three distinct Zn binding sites named A, B, and C. In this study, we use cryo-EM, microscale thermophoresis and molecular dynamics simulations to address the structural and functional roles of individual sites as well as the interplay between Zn binding and protonation. Structural studies indicate that site C in the cytoplasmic domain is primarily responsible for stabilizing the dimer and that site B at the cytoplasmic membrane surface controls the structural transition from an inward facing conformation to an occluded conformation. Binding data show that intramembrane site A, which is directly responsible for transport, has a dramatic pH dependence consistent with coupling to the proton motive force. A comprehensive thermodynamic model encompassing Zn binding and protonation states of individual residues indicates a transport stoichiometry of 1 Zn to 2-3 H depending on the external pH. This stoichiometry would be favorable in a physiological context, allowing the cell to use the proton gradient as well as the membrane potential to drive the export of Zn. | |||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 240.6 KB | Display | ![]() |
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PDB format | ![]() | 192.7 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 28885MC ![]() 8f6eC ![]() 8f6fC ![]() 8f6hC ![]() 8f6iC ![]() 8f6kC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 32441.203 Da / Num. of mol.: 2 / Mutation: D287A Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #2: Antibody | Mass: 23580.242 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #3: Antibody | Mass: 25406.352 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #4: Chemical | ChemComp-ZN / Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: D287A Zinc-loaded YiiP-Fab complex / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES | |||||||||||||||||||||||||
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Molecular weight | Value: 0.163024 MDa / Experimental value: NO | |||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 7.5 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||
Specimen support | Details: PELCO easiGLOW / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 700 nm / Alignment procedure: ZEMLIN TABLEAU |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2.5 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3686 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Sampling size: 5 µm / Width: 5760 / Height: 4092 |
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Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 4154002 / Details: template picking | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 252599 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 150.9 / Protocol: BACKBONE TRACE / Space: REAL / Target criteria: cross-correlation | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 7KZZ Accession code: 7KZZ / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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