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- EMDB-28886: Cryo-EM structure of a Zinc-loaded H263A/D287A mutant of the YiiP... -

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Basic information

Entry
Database: EMDB / ID: EMD-28886
TitleCryo-EM structure of a Zinc-loaded H263A/D287A mutant of the YiiP-Fab complex
Map data
Sample
  • Complex: D287A_H263A Zinc-loaded YiiP-Fab complex
    • Protein or peptide: Cadmium and zinc efflux pump FieF
  • 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.46 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)1R35GM144109 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM125081 United States
CitationJournal: To Be Published
Title: Characterization of individual Zn2+ binding sites of YiiP
Authors: Lopez-Redondo ML / Hussein AK / Stokes DL
History
DepositionNov 16, 2022-
Header (metadata) releaseFeb 8, 2023-
Map releaseFeb 8, 2023-
UpdateFeb 8, 2023-
Current statusFeb 8, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28886.png

Downloads & links

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Map

FileDownload / File: emd_28886.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.079 Å
Density
Contour LevelBy AUTHOR: 0.241
Minimum - Maximum-0.88738877 - 1.2876866
Average (Standard dev.)0.0004803704 (±0.017239952)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 388.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28886_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_28886_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_28886_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : D287A_H263A Zinc-loaded YiiP-Fab complex

EntireName: D287A_H263A Zinc-loaded YiiP-Fab complex
Components
  • Complex: D287A_H263A Zinc-loaded YiiP-Fab complex
    • Protein or peptide: Cadmium and zinc efflux pump FieF
  • Ligand: ZINC ION

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Supramolecule #1: D287A_H263A Zinc-loaded YiiP-Fab complex

SupramoleculeName: D287A_H263A Zinc-loaded YiiP-Fab complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Shewanella oneidensis (bacteria)
Molecular weightTheoretical: 325.261 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: 4 / Enantiomer: LEVO
Source (natural)Organism: Shewanella oneidensis (bacteria)
Molecular weightTheoretical: 32.374137 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 SLNEAASITD TTGLRVKAAF EDAEVIIHQA PVQVEPTTQ

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 4 / 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 chloridesodium chloride
20.0 millimolarC8H18N2O4SHEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)
2.0 mg/mlC22H42O11n-decyl-beta-D-maltoside
0.5 miilimolarZnSO4Zinc 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: 7.0 nm / 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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: 4113 / Average exposure time: 2.5 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3634729
Startup modelType of model: OTHER / Details: Ab Initio
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 227700 / Software - Name: cryoSPARC (ver. 3.1) / Details: Hetero-refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1) / Details: Non-uniform refinement
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 300844
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7kzz

RefinementSpace: REAL / Protocol: BACKBONE TRACE / Overall B value: 135.2 / Target criteria: cross-correlation
Output model

PDB-8f6k:
Cryo-EM structure of a Zinc-loaded H263A/D287A mutant of the YiiP-Fab complex

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