TY - JOUR
T1 - X-ray structure analysis of a metalloprotein with enhanced active-site resolution using in situ x-ray absorption near edge structure spectroscopy.
AU - Arcovito, Alessandro
AU - Benfatto, Maurizio
AU - Cianci, Michele
AU - Hasnain, Samar
AU - Nienhaus, Karin
AU - Nienhaus, Ulrich
AU - Savino, Carmelinda
AU - Strange, Richard
AU - Vallone, Beatrice
AU - Della Longa, Stefano
PY - 2007
Y1 - 2007
N2 - X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-Å resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with ±(0.02-0.07)-Å accuracy on the atomic distances and ±7° on the Fe-CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone.
AB - X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-Å resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with ±(0.02-0.07)-Å accuracy on the atomic distances and ±7° on the Fe-CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone.
KW - Crystallographic restraints
KW - Myoglobin
KW - Protein crystallography
KW - Synchroton radiation
KW - Crystallographic restraints
KW - Myoglobin
KW - Protein crystallography
KW - Synchroton radiation
UR - http://hdl.handle.net/10807/6795
M3 - Article
SN - 0027-8424
SP - 6211
EP - 6216
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
ER -