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- EMDB-5450: Inward-Facing Conformation of the Zinc Transporter YiiP revealed ... -

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Basic information

Entry
Database: EMDB / ID: EMD-5450
TitleInward-Facing Conformation of the Zinc Transporter YiiP revealed by Cryo-electron Microscopy
Map dataThis is one unit cell masked from YiiP tubular crystals imaged by cryo-EM
Sample
  • Sample: YiiP from Shewanella oneidensis in DOPG lipids
  • Protein or peptide: YiiP
KeywordsZinc transporter / transmembrane protein / electron crystallography / secondary transporter / alternating access mechanism
Function / homology
Function and homology information


zinc efflux active transmembrane transporter activity / cadmium ion transmembrane transporter activity / zinc ion transport / ferrous iron transmembrane transporter activity / intracellular zinc ion homeostasis / membrane => GO:0016020 / metal ion binding / plasma membrane
Similarity search - Function
Cation efflux protein, cytoplasmic domain / Dimerisation domain of Zinc Transporter / Cation efflux protein, cytoplasmic domain superfamily / Cation efflux protein / Cation efflux protein / Cation efflux transmembrane domain superfamily / Cation efflux family
Similarity search - Domain/homology
Cation-efflux pump FieF
Similarity search - Component
Biological speciesShewanella oneidensis MR-1 (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 13.0 Å
AuthorsCoudray N / Valvo S / Hu M / Lasala R / Kim C / Vink M / Zhou M / Provasi D / Filizola M / Tao J ...Coudray N / Valvo S / Hu M / Lasala R / Kim C / Vink M / Zhou M / Provasi D / Filizola M / Tao J / Fang J / Penczek PA / Ubarretxena-Belandia I / Stokes DL
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy.
Authors: Nicolas Coudray / Salvatore Valvo / Minghui Hu / Ralph Lasala / Changki Kim / Martin Vink / Ming Zhou / Davide Provasi / Marta Filizola / Juoehi Tao / Jia Fang / Pawel A Penczek / Iban ...Authors: Nicolas Coudray / Salvatore Valvo / Minghui Hu / Ralph Lasala / Changki Kim / Martin Vink / Ming Zhou / Davide Provasi / Marta Filizola / Juoehi Tao / Jia Fang / Pawel A Penczek / Iban Ubarretxena-Belandia / David L Stokes /
Abstract: YiiP is a dimeric Zn(2+)/H(+) antiporter from Escherichia coli belonging to the cation diffusion facilitator family. We used cryoelectron microscopy to determine a 13-Å resolution structure of a ...YiiP is a dimeric Zn(2+)/H(+) antiporter from Escherichia coli belonging to the cation diffusion facilitator family. We used cryoelectron microscopy to determine a 13-Å resolution structure of a YiiP homolog from Shewanella oneidensis within a lipid bilayer in the absence of Zn(2+). Starting from the X-ray structure in the presence of Zn(2+), we used molecular dynamics flexible fitting to build a model consistent with our map. Comparison of the structures suggests a conformational change that involves pivoting of a transmembrane, four-helix bundle (M1, M2, M4, and M5) relative to the M3-M6 helix pair. Although accessibility of transport sites in the X-ray model indicates that it represents an outward-facing state, our model is consistent with an inward-facing state, suggesting that the conformational change is relevant to the alternating access mechanism for transport. Molecular dynamics simulation of YiiP in a lipid environment was used to address the feasibility of this conformational change. Association of the C-terminal domains is the same in both states, and we speculate that this association is responsible for stabilizing the dimer that, in turn, may coordinate the rearrangement of the transmembrane helices.
History
DepositionJul 24, 2012-
Header (metadata) releaseSep 5, 2012-
Map releaseOct 10, 2012-
UpdateMar 2, 2016-
Current statusMar 2, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.33
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 3.33
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j1z
  • Surface level: 3.33
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5450_1.png

Downloads & links

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Map

FileDownload / File: emd_5450.map.gz / Format: CCP4 / Size: 19.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is one unit cell masked from YiiP tubular crystals imaged by cryo-EM
Voxel sizeX=Y=Z: 2.735 Å
Density
Contour LevelBy AUTHOR: 3.33 / Movie #1: 3.33
Minimum - Maximum-4.09629488 - 14.8743
Average (Standard dev.)0.00679428 (±0.22868274)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-80-80
Dimensions200160160
Spacing200160160
CellA: 437.59998 Å / B: 547.0 Å / C: 437.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.7352.7352.735
M x/y/z160200160
origin x/y/z0.0000.0000.000
length x/y/z437.600547.000437.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-80-100-80
NC/NR/NS160200160
D min/max/mean-4.09614.8740.007

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Supplemental data

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Sample components

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Entire : YiiP from Shewanella oneidensis in DOPG lipids

EntireName: YiiP from Shewanella oneidensis in DOPG lipids
Components
  • Sample: YiiP from Shewanella oneidensis in DOPG lipids
  • Protein or peptide: YiiP

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Supramolecule #1000: YiiP from Shewanella oneidensis in DOPG lipids

SupramoleculeName: YiiP from Shewanella oneidensis in DOPG lipids / type: sample / ID: 1000 / Oligomeric state: homodimer / Number unique components: 1
Molecular weightTheoretical: 33 KDa

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Macromolecule #1: YiiP

MacromoleculeName: YiiP / type: protein_or_peptide / ID: 1 / Name.synonym: Ferrous-iron efflux pump (FieF) / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Shewanella oneidensis MR-1 (bacteria) / Location in cell: Inner membrane
Molecular weightTheoretical: 33 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET
SequenceGO: zinc ion transport, membrane => GO:0016020 / InterPro: Cation efflux protein

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7 / Details: 20mM TES, 5mM MgCl2, 100mM NaCl, 5mM NaN3
GridDetails: Holey carbon grids
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 2-5 seconds before plunging.
DetailsProteins solubilized in DM were mixed with 18:1 dioleoylphosphatidyl glycerol lipids solubilized in DDM at a lipid-to-protein mass ratio of 1. This solution was dialyzed at room temperature for two weeks. Crystal cell parameters were a=57.5, b=34.0, c=100.0, alpha=90, beta=90, gamma=85.3.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 51190 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 100 K
Alignment procedureLegacy - Astigmatism: Objective astigmatism corrected at 200,000 times magnification
DateFeb 10, 2009
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 19 / Average electron dose: 10 e/Å2 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTF to the whole micrograph
Final reconstructionApplied symmetry - Helical parameters - Δz: 17.1 Å
Applied symmetry - Helical parameters - Δ&Phi: 56.4 °
Applied symmetry - Helical parameters - Axial symmetry: C3 (3 fold cyclic)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPARX, EMIP
DetailsIndexing of the helical symmetry was done by comparing the positions of individual layer lines in Fourier space with the outer radius of the tube in real space. IHRSR method used to calculate the 3D structure.

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Atomic model buiding 1

Initial modelPDB ID:

3h90


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: C
DetailsProtocol: MDFF. An initial homology model of YiiP from S. oneidensis was built with MODELLER 9v7 using the X-ray crystal structure of YiiP from E. coli (PDB entry 3H90) as a template. This model included 9 residues at the N-terminus and 4 residues at the C-terminus that were not present in the X-ray structure.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j1z:
Inward-Facing Conformation of the Zinc Transporter YiiP revealed by Cryo-electron Microscopy

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