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- EMDB-28884: Cryo-EM structure of a Zinc-loaded symmetrical D70A mutant of the... -

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Basic information

Entry
Database: EMDB / ID: EMD-28884
TitleCryo-EM structure of a Zinc-loaded symmetrical D70A mutant of the YiiP-Fab complex
Map data
Sample
  • Complex: D70A symmetrical Zinc-loaded YiiP-Fab complex
    • Protein or peptide: Cadmium and zinc efflux pump FieF
    • Protein or peptide: Fab2r light chain
    • Protein or peptide: Fab2r heavy chain
  • Ligand: ZINC ION
KeywordsZinc transporter / cation diffusion facilitator / membrane protein / transport protein
Function / homology
Function and homology information


zinc efflux antiporter 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) / Shewanella oneidensis MR-1 (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.03 Å
AuthorsLopez-Redondo ML / Hussein AK / Stokes DL
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM144109 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM125081 United States
CitationJournal: Elife / Year: 2023
Title: Energy coupling and stoichiometry of Zn/H antiport by the prokaryotic cation diffusion facilitator YiiP.
Authors: Adel Hussein / Shujie Fan / Maria Lopez-Redondo / Ian Kenney / Xihui Zhang / Oliver Beckstein / David L Stokes /
Abstract: YiiP from Shewanella oneidensis is a prokaryotic Zn/H antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for ...YiiP from Shewanella oneidensis is a prokaryotic Zn/H antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for homeostasis of transition metal ions. Previous studies of YiiP as well as related CDF transporters have established a homodimeric architecture and the presence of three distinct Zn binding sites named A, B, and C. In this study, we use cryo-EM, microscale thermophoresis and molecular dynamics simulations to address the structural and functional roles of individual sites as well as the interplay between Zn binding and protonation. Structural studies indicate that site C in the cytoplasmic domain is primarily responsible for stabilizing the dimer and that site B at the cytoplasmic membrane surface controls the structural transition from an inward facing conformation to an occluded conformation. Binding data show that intramembrane site A, which is directly responsible for transport, has a dramatic pH dependence consistent with coupling to the proton motive force. A comprehensive thermodynamic model encompassing Zn binding and protonation states of individual residues indicates a transport stoichiometry of 1 Zn to 2-3 H depending on the external pH. This stoichiometry would be favorable in a physiological context, allowing the cell to use the proton gradient as well as the membrane potential to drive the export of Zn.
History
DepositionNov 16, 2022-
Header (metadata) releaseFeb 8, 2023-
Map releaseFeb 8, 2023-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28884.png

Downloads & links

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Map

FileDownload / File: emd_28884.map.gz / Format: CCP4 / Size: 137.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 330 pix.
= 356.07 Å		1.08 Å/pix.
x 330 pix.
= 356.07 Å		1.08 Å/pix.
x 330 pix.
= 356.07 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.079 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.5267041 - 0.7421923
Average (Standard dev.)0.00018619784 (±0.012662138)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions330330330
Spacing330330330
CellA=B=C: 356.07 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28884_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_28884_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_28884_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : D70A symmetrical Zinc-loaded YiiP-Fab complex

EntireName: D70A symmetrical Zinc-loaded YiiP-Fab complex
Components
  • Complex: D70A symmetrical Zinc-loaded YiiP-Fab complex
    • Protein or peptide: Cadmium and zinc efflux pump FieF
    • Protein or peptide: Fab2r light chain
    • Protein or peptide: Fab2r heavy chain
  • Ligand: ZINC ION

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Supramolecule #1: D70A symmetrical Zinc-loaded YiiP-Fab complex

SupramoleculeName: D70A symmetrical Zinc-loaded YiiP-Fab complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Shewanella oneidensis (bacteria)
Molecular weightTheoretical: 163.024 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)
Molecular weightTheoretical: 32.441203 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTQTSQYDFW VKLASRASVA TALTLITIKL LAWLYSGSAS MLASLTDSFA DTLASIINFI AIRYAIVPAA HDHRYGHGKA EPLAALAQS AFIMGSAFLL LFYGGERLLN PSPVENATLG VVVSVVAIVL TLALVLLQKR ALAATNSTVV EADSLHYKSD L FLNAAVLL ...String:
MTQTSQYDFW VKLASRASVA TALTLITIKL LAWLYSGSAS MLASLTDSFA DTLASIINFI AIRYAIVPAA 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: Fab2r light chain

MacromoleculeName: Fab2r light chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.580242 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQIWSWPLIT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQIWSWPLIT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

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Macromolecule #3: Fab2r heavy chain

MacromoleculeName: Fab2r heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.406352 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF TIYSSSIHWV RQAPGKGLEW VASIYSSSGS TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARQSYSGLSP RRHWSYGAMD YWGQGTLVTV FNQIKGPSVF PLAPSSKSTS GGTAALGCLV K DYFPEPVT ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF TIYSSSIHWV RQAPGKGLEW VASIYSSSGS TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARQSYSGLSP RRHWSYGAMD YWGQGTLVTV FNQIKGPSVF PLAPSSKSTS GGTAALGCLV K DYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCDKTHT

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 millimolarNaClsodium chloride
20.0 millimolarC8H18N2O4SHEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)
2.0 mg/mlC22H42O11n-decyl-beta-D-maltoside
0.5 millimolarZnSO4zinc sulfate
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 7 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 5 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa / Details: PELCO easiGLOW
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 3420 / Average exposure time: 2.5 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1702119
Startup modelType of model: OTHER / Details: Ab Initio
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.03 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 182413
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1) / Details: Non-uniform refinement
Final 3D classificationNumber classes: 2 / Avg.num./class: 185000 / Software - Name: cryoSPARC (ver. 3.1) / Details: Hetero-refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7kzz


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: BACKBONE TRACE / Overall B value: 126 / Target criteria: cross-correlation
Output model

PDB-8f6i:
Cryo-EM structure of a Zinc-loaded symmetrical D70A mutant of the YiiP-Fab complex

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