TY - JOUR
T1 - Experimental phasing using zinc and sulfur anomalous signals measured at the zinc absorption peak
AU - Lee, Sangmin
AU - Kim, Min Kyu
AU - Ji, Chang Jun
AU - Lee, Jin Won
AU - Cha, Sun Shin
N1 - Funding Information:
This work was supported by the National Research Foundation of Korea Grant NRF-2012R1A2A2A02005978, the CAP through Korea Research Council of Fundamental Science Technology (KRCF), Korea Institute of Science and Technology (KIST), & Korea Institute of Ocean Science and Technology (KIOST), the Marine and Extreme Genome Research Center program, and the Development of Biohydrogen Production Technology Using Hyperthermophilic Archaea program of MOF. Works performed at Hanyang University were supported by the National Research Foundation of Korea Grant KRF-2008-313-C0077.
PY - 2013/10
Y1 - 2013/10
N2 - Iron is an essential transition metal required for bacterial growth and survival. Excess free iron can lead to the generation of reactive oxygen species that can cause severe damage to cellular functions. Cells have developed iron-sensing regulators to maintain iron homeostasis at the transcription level. The ferric uptake regulator (Fur) is an iron-responsive regulator that controls the expression of genes involved in iron homeostasis, bacterial virulence, stress resistance, and redox metabolism. Here, we report the expression, purification, crystallization, and phasing of the apo-form of Bacillus subtilis Fur (BsFur) in the absence of regulatory metal ions. Crystals were obtained by microbatch crystallization method at 295 K and diffraction data at a resolution of 2.6 Å was collected at the zinc peak wavelength (λ=1.2823 Å). Experimental phasing identified the positions of one zinc atom and four sulfur atoms of cysteine residues coordinating the zinc atom, indicating that the data contained a meaningful anomalous scattering originating from the ordered zinc-coordinating sulfur atoms, in spite of the small anomalous signals of sulfur atoms at the examined wavelength.
AB - Iron is an essential transition metal required for bacterial growth and survival. Excess free iron can lead to the generation of reactive oxygen species that can cause severe damage to cellular functions. Cells have developed iron-sensing regulators to maintain iron homeostasis at the transcription level. The ferric uptake regulator (Fur) is an iron-responsive regulator that controls the expression of genes involved in iron homeostasis, bacterial virulence, stress resistance, and redox metabolism. Here, we report the expression, purification, crystallization, and phasing of the apo-form of Bacillus subtilis Fur (BsFur) in the absence of regulatory metal ions. Crystals were obtained by microbatch crystallization method at 295 K and diffraction data at a resolution of 2.6 Å was collected at the zinc peak wavelength (λ=1.2823 Å). Experimental phasing identified the positions of one zinc atom and four sulfur atoms of cysteine residues coordinating the zinc atom, indicating that the data contained a meaningful anomalous scattering originating from the ordered zinc-coordinating sulfur atoms, in spite of the small anomalous signals of sulfur atoms at the examined wavelength.
KW - crystallization
KW - experimental phasing
KW - Ferric uptake regulator
KW - transcription regulator
UR - http://www.scopus.com/inward/record.url?scp=84887946844&partnerID=8YFLogxK
U2 - 10.1007/s12275-013-3412-2
DO - 10.1007/s12275-013-3412-2
M3 - Article
C2 - 24173644
AN - SCOPUS:84887946844
SN - 1225-8873
VL - 51
SP - 639
EP - 643
JO - Journal of Microbiology
JF - Journal of Microbiology
IS - 5
ER -