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- EMDB-23093: Cryo-EM structure of YiiP-Fab complex in Holo state -

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Basic information

Entry
Database: EMDB / ID: EMD-23093
TitleCryo-EM structure of YiiP-Fab complex in Holo state
Map datalocally sharpened map from cryosparc non-uniform refinement
Sample
  • Complex: YiiP_SO in complex with Fab2R
    • Complex: YiiP
      • Protein or peptide: Cadmium and zinc efflux pump FieF
    • Complex: Fab2r light chain
      • Protein or peptide: Fab2R light chain
    • Complex: Fab2r heavy chain
      • Protein or peptide: Fab2R heavy chain
  • Ligand: ZINC ION
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsLopez-Redondo ML / Fan S / Koide A / Koide S / Beckstein O / Stokes DL
Funding support United States, 1 items
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, 2020-
Header (metadata) releaseJan 6, 2021-
Map releaseJan 6, 2021-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kzz
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23093.png

Downloads & links

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Map

FileDownload / File: emd_23093.map.gz / Format: CCP4 / Size: 137.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocally sharpened map from cryosparc non-uniform refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 330 pix.
= 345.84 Å		1.05 Å/pix.
x 330 pix.
= 345.84 Å		1.05 Å/pix.
x 330 pix.
= 345.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.048 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6 / Movie #1: 0.6
Minimum - Maximum-2.487367 - 5.2021346
Average (Standard dev.)0.00040811513 (±0.074076)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions330330330
Spacing330330330
CellA=B=C: 345.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0481.0481.048
M x/y/z330330330
origin x/y/z0.0000.0000.000
length x/y/z345.840345.840345.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS330330330
D min/max/mean-2.4875.2020.000

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Supplemental data

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Mask #1

Fileemd_23093_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map from cryosparc non-uniform refinement

Fileemd_23093_half_map_1.map
Annotationhalf-map from cryosparc non-uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map from cryosparc non-uniform refinement

Fileemd_23093_half_map_2.map
Annotationhalf-map from cryosparc non-uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : YiiP_SO in complex with Fab2R

EntireName: YiiP_SO in complex with Fab2R
Components
  • Complex: YiiP_SO in complex with Fab2R
    • Complex: YiiP
      • Protein or peptide: Cadmium and zinc efflux pump FieF
    • Complex: Fab2r light chain
      • Protein or peptide: Fab2R light chain
    • Complex: Fab2r heavy chain
      • Protein or peptide: Fab2R heavy chain
  • Ligand: ZINC ION

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Supramolecule #1: YiiP_SO in complex with Fab2R

SupramoleculeName: YiiP_SO in complex with Fab2R / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: YiiP-Fab complex purified in decyl maltopyranoside (DM) detergent
Molecular weightExperimental: 25 KDa

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Supramolecule #2: YiiP

SupramoleculeName: YiiP / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Shewanella oneidensis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Fab2r light chain

SupramoleculeName: Fab2r light chain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #4: Fab2r heavy chain

SupramoleculeName: Fab2r heavy chain / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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 (bacteria)
Molecular weightTheoretical: 32.485211 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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

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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: 8 / 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

Concentration3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
150.0 mMNaClSodium Chloride
1.5 mg/mlDMdecyl maltopyranoside
1.0 mMTCEP(tris(2-carboxyethyl)phosphine)
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3 uL of protein mixture applied to grid. Blot time 4 seconds, blot force 0..
DetailsYiiP-Fab2R complex purified through SEC with DM, the same day as grid freezing. Main Peak fraction was used for freezing.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 77.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 15 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Number real images: 3674 / Average exposure time: 9.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
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: 1448191
CTF correctionSoftware - Name: cryoSPARC (ver. 2.9.0) / Software - details: patch ctf
Startup modelType of model: NONE / Details: reference-free alignment
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.9.0.) / Software - details: non-linear refinement / Number images used: 151898
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2.9.0.) / Software - details: ab initio
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9.0) / Software - details: non-linear refinement
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 2.9.0) / Software - details: heterogeneous refinement / Details: Heterogenous Refinement.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5vrf

RefinementSpace: REAL / Protocol: BACKBONE TRACE / Overall B value: 76 / Target criteria: Correlation coefficient
Output model

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

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