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- PDB-4arh: X ray structure of the periplasmic zinc binding protein ZinT from... -

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Basic information

Entry
Database: PDB / ID: 4arh
TitleX ray structure of the periplasmic zinc binding protein ZinT from Salmonella enterica
ComponentsMETAL-BINDING PROTEIN YODA
KeywordsMETAL BINDING PROTEIN / PERIPLASMIC PROTEIN
Function / homologyCalycin beta-barrel core domain / Lipocalin / Beta Barrel / Mainly Beta / :
Function and homology information
Biological speciesSALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 Å
AuthorsAlaleona, F. / Ilari, A. / Battistoni, A. / Petrarca, P. / Chiancone, E.
CitationJournal: Biochim Biophys Acta / Year: 2014
Title: The Salmonella enterica ZinT structure, zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc.
Authors: Andrea Ilari / Flaminia Alaleona / Giancarlo Tria / Patrizia Petrarca / Andrea Battistoni / Carlotta Zamparelli / Daniela Verzili / Mattia Falconi / Emilia Chiancone /
Abstract: BACKGROUND: In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the ...BACKGROUND: In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT-ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim.
METHODS: ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy).
RESULTS: The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with ...RESULTS: The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a KD value of 22±2nM for ZinT, while those with ZnuA point to one high affinity (KD<20nM) and one low affinity Zn(II) binding site (KD in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT-ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other.
CONCLUSIONS: ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA.
GENERAL SIGNIFICANCE: Knowledge of the ZinT-ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.
History
DepositionApr 24, 2012Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 8, 2013Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2013Group: Database references
Revision 1.2May 1, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: METAL-BINDING PROTEIN YODA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,0727
Polymers21,4951
Non-polymers5766
Water2,450136
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)58.534, 58.534, 289.316
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number178
Space group name H-MP6122

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Components

#1: Protein METAL-BINDING PROTEIN YODA / ZINT


Mass: 21495.193 Da / Num. of mol.: 1 / Fragment: RESIDUES 31-216
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR (bacteria)
Strain: CMV23701 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): DH5[ALPHA] / References: UniProt: B5MZR0
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: SO4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 136 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.41 Å3/Da / Density % sol: 63.92 % / Description: NONE
Crystal growpH: 5 / Details: AMMONIUM SULFATE 2 M, SODIUM ACETATE 0.1 M PH 5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.918
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Jul 22, 2010
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.918 Å / Relative weight: 1
ReflectionResolution: 2.3→50 Å / Num. obs: 14007 / % possible obs: 99.1 % / Observed criterion σ(I): 0 / Redundancy: 12.7 % / Rmerge(I) obs: 0.11 / Net I/σ(I): 25.62
Reflection shellResolution: 2.3→2.38 Å / Redundancy: 14.3 % / Rmerge(I) obs: 0.24 / Mean I/σ(I) obs: 14.75 / % possible all: 99.9

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Processing

Software
NameVersionClassification
REFMAC5.5.0070refinement
DENZOdata reduction
SCALEPACKdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 10EK

10ek


Resolution: 2.3→50.69 Å / Cor.coef. Fo:Fc: 0.919 / Cor.coef. Fo:Fc free: 0.852 / SU B: 6.672 / SU ML: 0.165 / Cross valid method: THROUGHOUT / ESU R: 0.271 / ESU R Free: 0.245 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.
RfactorNum. reflection% reflectionSelection details
Rfree0.28841 693 5 %RANDOM
Rwork0.22054 ---
obs0.22388 13233 99.26 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 23.858 Å2
Baniso -1Baniso -2Baniso -3
1--0.05 Å2-0.03 Å20 Å2
2---0.05 Å20 Å2
3---0.08 Å2
Refinement stepCycle: LAST / Resolution: 2.3→50.69 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1503 0 30 136 1669
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.0221641
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.2831.9562240
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.5475207
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.35824.82887
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.53715283
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.068158
X-RAY DIFFRACTIONr_chiral_restr0.0920.2226
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.0211276
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.7561.5960
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it1.45721556
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it1.9933681
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it3.3654.5673
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.302→2.362 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.386 57 -
Rwork0.282 937 -
obs--99.8 %

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