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

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Basic information

Entry
Database: PDB / ID: 8f6i
TitleCryo-EM structure of a Zinc-loaded symmetrical D70A mutant of the YiiP-Fab complex
Components
  • Cadmium and zinc efflux pump FieF
  • Fab2r heavy chain
  • Fab2r light chain
KeywordsTRANSPORT PROTEIN / Zinc transporter / cation diffusion facilitator / membrane protein
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)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.03 Å
AuthorsLopez-Redondo, M.L. / Hussein, A.K. / Stokes, D.L.
Funding support United States, 2items
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: To Be Published
Title: Characterization of individual Zn2+ binding sites of YiiP
Authors: Lopez-Redondo, M.L. / Hussein, A.K. / Stokes, D.L.
History
DepositionNov 16, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 8, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Cadmium and zinc efflux pump FieF
A: Cadmium and zinc efflux pump FieF
E: Fab2r light chain
F: Fab2r heavy chain
C: Fab2r light chain
D: Fab2r heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,24812
Polymers162,8566
Non-polymers3926
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Cadmium and zinc efflux pump FieF


Mass: 32441.203 Da / Num. of mol.: 2 / Mutation: D70A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shewanella oneidensis MR-1 (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.: 6 / 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

ComponentName: D70A symmetrical Zinc-loaded YiiP-Fab complex / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.163024 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Shewanella oneidensis (bacteria)70863
31Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
220 mMHEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)C8H18N2O4S1
32 mg/mln-decyl-beta-D-maltosideC22H42O111
40.5 mMzinc sulfateZnSO41
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: PELCO easiGLOW / Grid 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: 277.15 K

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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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 700 nm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3420
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARC3.1CTF correction
7UCSF Chimera1.14model fitting
9PHENIX1.18model refinement
10cryoSPARC3.1initial Euler assignment
11cryoSPARC3.1final Euler assignment
12cryoSPARC3.1classification
13cryoSPARC3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1702119
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 182413 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 126 / Protocol: BACKBONE TRACE / Space: REAL / Target criteria: cross-correlation
Atomic model buildingPDB-ID: 7KZZ

7kzz

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00210595
ELECTRON MICROSCOPYf_angle_d0.50514425
ELECTRON MICROSCOPYf_dihedral_angle_d3.7451456
ELECTRON MICROSCOPYf_chiral_restr0.0391678
ELECTRON MICROSCOPYf_plane_restr0.0031818

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