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

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Basic information

Entry
Database: PDB / ID: 5vrf
TitleCryoEM Structure of the Zinc Transporter YiiP from helical crystals
ComponentsCadmium and zinc efflux pump FieF
KeywordsMEMBRANE PROTEIN / zinc antiporter / 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)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsCoudray, N. / Lopez-Redondo, M. / Zhang, Z. / Alexopoulos, J. / Stokes, D.L. / Transcontinental EM Initiative for Membrane Protein Structure (TEMIMPS)
Funding support United States, 2items
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, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 14, 2018Provider: repository / Type: Initial release
Revision 1.1Mar 28, 2018Group: Data collection / Database references / Other / Category: cell / citation
Item: _cell.Z_PDB / _cell.length_a ..._cell.Z_PDB / _cell.length_a / _cell.length_b / _cell.length_c / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 23, 2019Group: Data collection / Refinement description / Category: em_3d_fitting_list
Revision 1.3Feb 20, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization
Revision 1.5Mar 13, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

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
Polymers64,9702
Non-polymers5238
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5350 Å2
ΔGint-249 kcal/mol
Surface area31850 Å2
MethodPISA

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Components

#1: Protein Cadmium and zinc efflux pump FieF


Mass: 32485.211 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shewanella oneidensis MR-1 (bacteria) / Strain: MR-1 / Gene: fieF, SO_4475 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 (DE3) / References: UniProt: Q8E919
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Formula: Zn

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: YiiP dimer in an inward-facing conformation / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.06 MDa / Experimental value: YES
Source (natural)Organism: Shewanella oneidensis MR-1 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21 (DE3)
Buffer solutionpH: 7 / Details: Buffer was changed twice per day.
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaClSodium chloride1
25 mMmagnesium chlorideMgCl21
35 mMsodium azideNaN31
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The protein was purified in 0.2% n-dodecyl beta-D-maltoside
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: EMS
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: LAB6 / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / 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) / Num. of grids imaged: 3 / Num. of real images: 2743
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 50 / Used frames/image: 2-16

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1SPARX/EMAN2EMAN2.1particle selectionsxhelixboxer.py
2RELION2particle selection2D Classissification
3Leginonimage acquisition
6CTFFIND4.1CTF correction
7Gctf0.5CTF correction
10Coot0.8.6model fitting
12RELION2initial Euler assignment
13RELION2final Euler assignment
14RELION2classification
15RELION23D reconstruction
16PHENIX1.11model refinementreal_space_refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -17 ° / Axial rise/subunit: 28.2 Å / Axial symmetry: D5
Particle selectionNum. of particles selected: 141904
Details: 2293 filaments selected with SPARX/EMAN2, then windowed into 141,904 segments (450x450 pixel overlapping segments)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 72333
Details: Reconstruction was achieved using the IHRSR method implemented in RELION 2.0.
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
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 ...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.
Atomic model building

3D fitting-ID: 1 / Accession code: 3J1Z / Initial refinement model-ID: 1 / Pdb chain residue range: 7-288 / PDB-ID: 3J1Z

3j1z

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B
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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