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- PDB-7kzz: Cryo-EM structure of YiiP-Fab complex in Holo state -

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Basic information

Entry
Database: PDB / ID: 7kzz
TitleCryo-EM structure of YiiP-Fab complex in Holo state
Components
  • Cadmium and zinc efflux pump FieF
  • Fab2R heavy chain
  • Fab2R light chain
KeywordsMEMBRANE PROTEIN / Zinc transport / cation diffusion facilitator / holo state / inward-facing state
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 (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsLopez-Redondo, M.L. / Fan, S. / Koide, A. / Koide, S. / Beckstein, O. / Stokes, D.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM125081 United States
CitationJournal: J Gen Physiol / Year: 2021
Title: Zinc binding alters the conformational dynamics and drives the transport cycle of the cation diffusion facilitator YiiP.
Authors: Maria Lopez-Redondo / Shujie Fan / Akiko Koide / Shohei Koide / Oliver Beckstein / David L Stokes /
Abstract: YiiP is a secondary transporter that couples Zn2+ transport to the proton motive force. Structural studies of YiiP from prokaryotes and Znt8 from humans have revealed three different Zn2+ sites and a ...YiiP is a secondary transporter that couples Zn2+ transport to the proton motive force. Structural studies of YiiP from prokaryotes and Znt8 from humans have revealed three different Zn2+ sites and a conserved homodimeric architecture. These structures define the inward-facing and outward-facing states that characterize the archetypal alternating access mechanism of transport. To study the effects of Zn2+ binding on the conformational transition, we use cryo-EM together with molecular dynamics simulation to compare structures of YiiP from Shewanella oneidensis in the presence and absence of Zn2+. To enable single-particle cryo-EM, we used a phage-display library to develop a Fab antibody fragment with high affinity for YiiP, thus producing a YiiP/Fab complex. To perform MD simulations, we developed a nonbonded dummy model for Zn2+ and validated its performance with known Zn2+-binding proteins. Using these tools, we find that, in the presence of Zn2+, YiiP adopts an inward-facing conformation consistent with that previously seen in tubular crystals. After removal of Zn2+ with high-affinity chelators, YiiP exhibits enhanced flexibility and adopts a novel conformation that appears to be intermediate between inward-facing and outward-facing states. This conformation involves closure of a hydrophobic gate that has been postulated to control access to the primary transport site. Comparison of several independent cryo-EM maps suggests that the transition from the inward-facing state is controlled by occupancy of a secondary Zn2+ site at the cytoplasmic membrane interface. This work enhances our understanding of individual Zn2+ binding sites and their role in the conformational dynamics that govern the transport cycle.
History
DepositionDec 10, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 6, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 28, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
B: Cadmium and zinc efflux pump FieF
E: Fab2R light chain
F: Fab2R heavy chain
A: Cadmium and zinc efflux pump FieF
C: Fab2R light chain
D: Fab2R heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,46714
Polymers162,9446
Non-polymers5238
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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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 (bacteria) / Gene: fieF, SO_4475 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8E919
#2: Antibody Fab2R light chain


Mass: 23580.242 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody Fab2R heavy chain


Mass: 25406.352 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1YiiP_SO in complex with Fab2RCOMPLEXYiiP-Fab complex purified in decyl maltopyranoside (DM) detergent#1-#30MULTIPLE SOURCES
2YiiPCOMPLEX#11RECOMBINANT
3Fab2r light chainCOMPLEX#21RECOMBINANT
4Fab2r heavy chainCOMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.172 MDaNO
210.066 MDaYES
310.023 MDaYES
410.025 MDaYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Shewanella oneidensis (bacteria)70863
23Homo sapiens (human)9606
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES1
2150 mMSodium ChlorideNaCl1
31.5 mg/mldecyl maltopyranosideDM1
41 mM(tris(2-carboxyethyl)phosphine)TCEP1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: YiiP-Fab2R complex purified through SEC with DM, the same day as grid freezing. Main Peak fraction was used for freezing.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: 3 uL of protein mixture applied to grid. Blot time 4 seconds, blot force 0.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K
Image recordingAverage exposure time: 9 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3674
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 15 eV
Image scansSampling size: 14 µm / Width: 3710 / Height: 3838 / Movie frames/image: 45

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18_3845refinement
PHENIX1.18_3845refinement
EM software
IDNameVersionCategoryDetails
2Leginonimage acquisition
4cryoSPARC2.9.0CTF correctionpatch ctf
7UCSF Chimera1.13.1model fitting
9cryoSPARC2.9.0.initial Euler assignmentab initio
10cryoSPARC2.9.0final Euler assignmentnon-linear refinement
11cryoSPARC2.9.0classificationheterogeneous refinement
12cryoSPARC2.9.0.3D reconstructionnon-linear refinement
13PHENIX1.18model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1448191
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 151898 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 76 / Protocol: BACKBONE TRACE / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 5VRF

5vrf

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 81.71 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.014910688
ELECTRON MICROSCOPYf_angle_d2.418414554
ELECTRON MICROSCOPYf_chiral_restr0.13461694
ELECTRON MICROSCOPYf_plane_restr0.02381836
ELECTRON MICROSCOPYf_dihedral_angle_d14.85821470

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