|Entry||Database: PDB / ID: 5vrf|
|Title||CryoEM Structure of the Zinc Transporter YiiP from helical crystals|
|Components||Cadmium and zinc efflux pump FieF|
|Keywords||MEMBRANE PROTEIN / zinc antiporter / membrane protein / cation diffusion facilitator / metal transport / helical crystals / Structural Genomics / PSI-Biology / Transcontinental EM Initiative for Membrane Protein Structure / TEMIMPS|
|Function/homology||Cation efflux protein, cytoplasmic domain superfamily / Cation efflux protein, cytoplasmic domain / Cation efflux protein / Cation efflux transmembrane domain superfamily / Cation efflux family / Dimerisation domain of Zinc Transporter / cation transmembrane transporter activity / integral component of membrane / plasma membrane / Cadmium and zinc efflux pump FieF|
Function and homology information
|Specimen source||Shewanella oneidensis mr-1 / bacteria /|
|Method||Electron microscopy (4.1 Å resolution / Helical array / Helical) / Transmission electron microscopy|
|Authors||Coudray, N. / Lopez-Redondo, M. / Zhang, Z. / Alexopoulos, J. / Stokes, D.L. / Transcontinental EM Initiative for Membrane Protein Structure (TEMIMPS)|
|Citation||Journal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2018|
Title: Structural basis for the alternating access mechanism of the cation diffusion facilitator YiiP.
Authors: Maria Luisa Lopez-Redondo / Nicolas Coudray / Zhening Zhang / John Alexopoulos / David L Stokes
Abstract: YiiP is a dimeric antiporter from the cation diffusion facilitator family that uses the proton motive force to transport Zn across bacterial membranes. Previous work defined the atomic structure of ...YiiP is a dimeric antiporter from the cation diffusion facilitator family that uses the proton motive force to transport Zn across bacterial membranes. Previous work defined the atomic structure of an outward-facing conformation, the location of several Zn binding sites, and hydrophobic residues that appear to control access to the transport sites from the cytoplasm. A low-resolution cryo-EM structure revealed changes within the membrane domain that were associated with the alternating access mechanism for transport. In the current work, the resolution of this cryo-EM structure has been extended to 4.1 Å. Comparison with the X-ray structure defines the differences between inward-facing and outward-facing conformations at an atomic level. These differences include rocking and twisting of a four-helix bundle that harbors the Zn transport site and controls its accessibility within each monomer. As previously noted, membrane domains are closely associated in the dimeric structure from cryo-EM but dramatically splayed apart in the X-ray structure. Cysteine crosslinking was used to constrain these membrane domains and to show that this large-scale splaying was not necessary for transport activity. Furthermore, dimer stability was not compromised by mutagenesis of elements in the cytoplasmic domain, suggesting that the extensive interface between membrane domains is a strong determinant of dimerization. As with other secondary transporters, this interface could provide a stable scaffold for movements of the four-helix bundle that confers alternating access of these ions to opposite sides of the membrane.
SummaryFull reportAbout validation report
|Date||Deposition: May 10, 2017 / Release: Mar 14, 2018|
Downloads & links
B: Cadmium and zinc efflux pump FieF
A: Cadmium and zinc efflux pump FieF
Mass: 32485.211 Da / Num. of mol.: 2
Source: (gene. exp.) Shewanella oneidensis mr-1 / bacteria /
Strain: MR-1 / Gene: fieF, SO_4475 / Production host: Escherichia coli BL21(DE3) / Strain (production host): BL21 (DE3) / References: UniProt:Q8E919
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: HELICAL ARRAY / Reconstruction method: HELICAL|
|Component||Name: YiiP dimer in an inward-facing conformation / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT|
|Molecular weight||Value: 0.06 deg. / Units: MEGADALTONS / Experimental value: YES|
|Source (natural)||Organism: Shewanella oneidensis MR-1|
|Source (recombinant)||Organism: Escherichia coli BL21(DE3) / Strain: BL21 (DE3)|
|Buffer solution||Details: Buffer was changed twice per day. / pH: 7|
|Specimen||Conc.: 0.6 mg/ml|
Details: The protein was purified in 0.2% n-dodecyl beta-D-maltoside
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
|Specimen support||Grid material: COPPER / Grid mesh size: 400 / Grid type: EMS|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 %|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: LAB6 / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELD / Nominal magnification: 22500 / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 5400 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Average exposure time: 10 sec. / Electron dose: 70 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 3 / Number of real images: 2743|
|Image scans||Dimension width: 3710 / Dimension height: 3838 / Movie frames/image: 50 / Used frames/image: 2-16|
|Software||Name: PHENIX / Version: 1.11.1_2575: / Classification: refinement|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Helical symmerty||Angular rotation/subunit: -17 deg. / Axial rise/subunit: 28.2 Å / Axial symmetry: D5|
|Particle selection||Details: 2293 filaments selected with SPARX/EMAN2, then windowed into 141,904 segments (450x450 pixel overlapping segments)|
Number of particles selected: 141904
|3D reconstruction||Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 72333|
Details: Reconstruction was achieved using the IHRSR method implemented in RELION 2.0.
Symmetry type: HELICAL
|Atomic model building||Details: The residues were manually adjusted in COOT relying on the large side-chain residues, and the whole system was further optimized using the real_space_refine algorithm in Phenix to ensure proper fit. The conformation was subjected to 13 rounds of COOT/Phenix refinements.|
Ref protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: cross-correlation coefficient
|Atomic model building||PDB-ID: 3J1Z|
Pdb chain ID: A,B / Pdb chain residue range: 7-288
|Refine LS restraints|
-Jul 12, 2017. Major update of PDB
Major update of PDB
- wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary. This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated. See below links for details.
- In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software). Now, EM Navigator and Yorodumi are based on the updated data.
-Jun 16, 2017. Omokage search with filter
Omokage search with filter
- Result of Omokage search can be filtered by keywords and the database types
Related info.: Omokage search
+Sep 15, 2016. EM Navigator & Yorodumi renewed
EM Navigator & Yorodumi renewed
- New versions of EM Navigator and Yorodumi started
Related info.: Changes in new EM Navigator and Yorodumi / EM Navigator (legacy version) / Yorodumi (legacy version)
+Aug 31, 2016. New EM Navigator & Yorodumi
New EM Navigator & Yorodumi
- In 15th Sep 2016, the development versions of EM Navigator and Yorodumi will replace the official versions.
- Current version will continue as 'legacy version' for some time.
Related info.: Changes in new EM Navigator and Yorodumi / EM Navigator / Yorodumi / EM Navigator (legacy version) / Yorodumi (legacy version)
+Apr 13, 2016. Omokage search got faster
Omokage search got faster
- The computation time became ~1/2 compared to the previous version by re-optimization of data accession
- Enjoy "shape similarity" of biomolecules, more!
Related info.: Omokage search
Thousand views of thousand structures
- Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
- All the functionalities will be ported from the levgacy version.
- This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
Related info.: Yorodumi (legacy version) / EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi