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- EMDB-8728: CryoEM Structure of the Zinc Transporter YiiP from helical crystals -

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Basic information

Entry
Database: EMDB / ID: EMD-8728
TitleCryoEM Structure of the Zinc Transporter YiiP from helical crystals
Map dataprimary map
Sample
  • Complex: YiiP dimer in an inward-facing conformation
    • Protein or peptide: Cadmium and zinc efflux pump FieF
  • Ligand: ZINC ION
Keywordszinc antiporter / membrane protein / cation diffusion facilitator / metal transport / helical crystals / Structural Genomics / PSI-Biology / Transcontinental EM Initiative for Membrane Protein Structure / TEMIMPS
Function / homology
Function and homology information


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
Cation efflux protein, cytoplasmic domain / Dimerisation domain of Zinc Transporter / Cation efflux protein, cytoplasmic domain superfamily / 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: 4.1 Å
AuthorsCoudray N
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U54 GM94598 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM095747 United States
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.
History
DepositionMay 10, 2017-
Header (metadata) releaseJul 26, 2017-
Map releaseMar 14, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5vrf
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8728.png

Downloads & links

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Map

FileDownload / File: emd_8728.map.gz / Format: CCP4 / Size: 8.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprimary map
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.018 / Movie #1: 0.018
Minimum - Maximum-0.035158023 - 0.075662784
Average (Standard dev.)0.0005571464 (±0.003660637)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions131131131
Spacing131131131
CellA=B=C: 140.17001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z131131131
origin x/y/z0.0000.0000.000
length x/y/z140.170140.170140.170
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS131131131
D min/max/mean-0.0350.0760.001

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Supplemental data

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Sample components

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Entire : YiiP dimer in an inward-facing conformation

EntireName: YiiP dimer in an inward-facing conformation
Components
  • Complex: YiiP dimer in an inward-facing conformation
    • Protein or peptide: Cadmium and zinc efflux pump FieF
  • Ligand: ZINC ION

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Supramolecule #1: YiiP dimer in an inward-facing conformation

SupramoleculeName: YiiP dimer in an inward-facing conformation / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Shewanella oneidensis MR-1 (bacteria)
Molecular weightTheoretical: 60 KDa

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Macromolecule #1: Cadmium and zinc efflux pump FieF

MacromoleculeName: Cadmium and zinc efflux pump FieF / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Shewanella oneidensis MR-1 (bacteria) / Strain: MR-1
Molecular weightTheoretical: 32.485211 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTQTSQYDFW VKLASRASVA TALTLITIKL LAWLYSGSAS MLASLTDSFA DTLASIINFI AIRYAIVPAD HDHRYGHGKA EPLAALAQS AFIMGSAFLL LFYGGERLLN PSPVENATLG VVVSVVAIVL TLALVLLQKR ALAATNSTVV EADSLHYKSD L FLNAAVLL ...String:
MTQTSQYDFW VKLASRASVA TALTLITIKL LAWLYSGSAS MLASLTDSFA DTLASIINFI AIRYAIVPAD HDHRYGHGKA EPLAALAQS AFIMGSAFLL LFYGGERLLN PSPVENATLG VVVSVVAIVL TLALVLLQKR ALAATNSTVV EADSLHYKSD L FLNAAVLL ALVLSQYGWW WADGLFAVLI ACYIGQQAFD LGYRSIQALL DRELDEDTRQ RIKLIAKEDP RVLGLHDLRT RQ AGKTVFI QFHLELDGNL SLNEAHSITD TTGLRVKAAF EDAEVIIHQD PVQVEPTTQ

UniProtKB: Cation-efflux pump FieF

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 8 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration0.6 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chloridesodium chloride
5.0 mMMgCl2magnesium chloride
5.0 mMNaN3sodium azide

Details: Buffer was changed twice per day.
GridModel: EMS / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: HOMEMADE PLUNGER
DetailsThe protein was purified in 0.2% n-dodecyl beta-D-maltoside

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: LAB6
Electron opticsCalibrated defocus max: 5.4 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 2-16 / Number grids imaged: 3 / Number real images: 2743 / Average exposure time: 10.0 sec. / Average electron dose: 70.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 141904
Software:
Namedetails
SPARX/EMAN2 (ver. EMAN2.1)sxhelixboxer.py
RELION (ver. 2.0)2D Classissification

Details: 2293 filaments selected with SPARX/EMAN2, then windowed into 141,904 segments (450x450 pixel overlapping segments)
Startup modelType of model: OTHER
Details: A map was obtained previously with images acquired on a JEOL2100 microscope. This map was used as a startup model (That map was obtained as follow: we started with a noisy cylinder. Then, a ...Details: A map was obtained previously with images acquired on a JEOL2100 microscope. This map was used as a startup model (That map was obtained as follow: we started with a noisy cylinder. Then, a first reconstruction was obtained with Helicon (from SPARX) and refined with Frealix).
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 2.0)
Final reconstructionApplied symmetry - Helical parameters - Δz: 28.2 Å
Applied symmetry - Helical parameters - Δ&Phi: -17.0 °
Applied symmetry - Helical parameters - Axial symmetry: D5 (2x5 fold dihedral)
Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0)
Details: Reconstruction was achieved using the IHRSR method implemented in RELION 2.0.
Number images used: 72333
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

3j1z

chain_id: A, residue_range: 7-288, source_name: PDB, initial_model_type: experimental model

3j1z

chain_id: B, residue_range: 7-288, source_name: PDB, initial_model_type: experimental model
DetailsThe 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross-correlation coefficient
Output model

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

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