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- PDB-5vrf: CryoEM Structure of the Zinc Transporter YiiP from helical crystals -

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Basic information

Database: PDB / ID: 5vrf
TitleCryoEM Structure of the Zinc Transporter YiiP from helical crystals
ComponentsCadmium and zinc efflux pump FieF
KeywordsMEMBRANE 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/homologyCation 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 sourceShewanella oneidensis mr-1 / bacteria /
MethodElectron microscopy (4.1 Å resolution / Helical array / Helical) / Transmission electron microscopy
AuthorsCoudray, N. / Lopez-Redondo, M. / Zhang, Z. / Alexopoulos, J. / Stokes, D.L. / Transcontinental EM Initiative for Membrane Protein Structure (TEMIMPS)
CitationJournal: 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: May 10, 2017 / Release: Mar 14, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Mar 14, 2018Structure modelrepositoryInitial release
1.1Mar 28, 2018Structure modelData collection / Database references / Othercell / citation_cell.Z_PDB / _cell.length_a / _cell.length_b / _cell.length_c / _citation.journal_volume / _citation.page_first / _citation.page_last

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Deposited unit
B: Cadmium and zinc efflux pump FieF
A: Cadmium and zinc efflux pump FieF
hetero molecules

Theoretical massNumber of molelcules
Total (without water)65,49410

TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)5350
ΔGint (kcal/M)-249
Surface area (Å2)31850


#1: Protein/peptide 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
#2: Chemical
ChemComp-ZN / ZINC ION

Mass: 65.409 Da / Num. of mol.: 8 / Formula: Zn / : Zinc

Experimental details


EM experimentAggregation state: HELICAL ARRAY / Reconstruction method: HELICAL

Sample preparation

ComponentName: YiiP dimer in an inward-facing conformation / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 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 solutionDetails: Buffer was changed twice per day. / pH: 7
Buffer component
IDConc.UnitsNameFormulaBuffer ID
1100mMsodium chlorideNaCl1
25mMmagnesium chlorideMgCl21
35mMsodium azideNaN31
SpecimenConc.: 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 supportGrid material: COPPER / Grid mesh size: 400 / Grid type: EMS
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 %

Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: LAB6 / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 scansDimension width: 3710 / Dimension height: 3838 / Movie frames/image: 50 / Used frames/image: 2-16


SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
16PHENIX1.11MODEL REFINEMENTreal_space_refine
Helical symmertyAngular rotation/subunit: -17 deg. / Axial rise/subunit: 28.2 Å / Axial symmetry: D5
Particle selectionDetails: 2293 filaments selected with SPARX/EMAN2, then windowed into 141,904 segments (450x450 pixel overlapping segments)
Number of particles selected: 141904
3D reconstructionResolution: 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 buildingDetails: 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 buildingPDB-ID: 3J1Z
Pdb chain ID: A,B / Pdb chain residue range: 7-288
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0114458
ELECTRON MICROSCOPYf_angle_d1.2746074
ELECTRON MICROSCOPYf_dihedral_angle_d3.7652622
ELECTRON MICROSCOPYf_chiral_restr0.056730
ELECTRON MICROSCOPYf_plane_restr0.008760

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