Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting method

Abstract

Synopsis A novel protein structure was solved by S-SAD using in-house chromium radiation. To reduce X-ray absorption of the cryo-buffer and cryoloop, a novel free crystal mounting technique was developed.Abstract A novel and easy crystal mounting technique was developed for the sulphur SAD method using chromium Kα radiation (2.29Å). Using this technique, the cryo-buffer and cryoloop around the protein crystal can be removed before data collection to eliminate their X-ray absorption. The superiority and the reproducibility of the datasets with our mounting technique were demonstrated using tetragonal hen egg-white lysozyme crystals. The structure of a novel protein, PH1109, from Pyrococcus horikoshii OT3 was solved using this technique. At the wavelength of chromium Kα radiation, anomalous signals, <|ΔF|>/<|F|> of PH1109 is expected to be 1.72%, as this protein of 144 residues includes 4 methionines and 2 cysteines. Sulphur SAD phasing was performed using SHELXD and SHELXE. In the case of the dataset obtained using our novel crystal mounting technique, 54.9 % of all residues were built with side-chains automatically by RESOLVE. On the other hand, only 16.0 % were built with side-chains for the dataset collected using the standard cryoloop. These results indicated that our crystal mounting technique was superior to the standard loop mounting method for measurement of small anomalous differences at longer wavelength, and yielded better results in sulphur substructure solution and initial phasing. The present study demonstrated that the sulphur SAD method with a chromium source becomes enhanced more practical for macromolecular structure determination using our crystal mounting technique.

Structural and Functional Analysis of a Glycoside Hydrolase Family 97 Enzyme from Bacteroides thetaiotaomicron

“…The crystal was transferred to reservoir solution supplemented with 35% (v/v) glycerol as a cryoprotectant, and then mounted using a loop-and buffer-less mount method (18) and flash-cooled under a stream of nitrogen gas at 100 K.…”

Anomalous diffraction in crystallographic phase evaluation

“…The growing successes of SAD spurred a renewed interest in home-source experiments (Yang & Pflugrath, 2001; Nagem et al, 2005), notably employing x-rays generated from a chromium anode to better exploit sulfur anomalous scattering (Yang et al, 2003; Kitago et al, 2005) or to take advantage of the anomalous scattering from iodine as its L I edge is approached (Evans & Bricogne, 2003). Nevertheless, synchrotron radiation provides compelling advantages; fine tuning of x-ray energy makes it possible to optimize the strength of f″ at the Se K-edge and other resonance peaks, and access to lower x-ray energies permits the enhancement of anomalous signals from low-Z elements such as sulfur since f″ values increase as these K-edges are approached (Weiss et al, 2001).…”

Lab Source Anomalous Scattering Using Cr Kα Radiation

“…SAD phasing has been carried out earlier on proteins like HEWL, GI and TL using synchrotron beamlines and in-house copper wavelengths [27,28,31]. Using in-house chromium X-ray source, HEWL structure was attempted using different mounting techniques [29]. Using in-house chromium wavelength, glucose isomerase structure was also attempted using different solving techniques like ARP/wARP and OASIS2004 [30], but the complete structure could not be solved.…”

“…Most of the sulfur SAD data have been collected on tunable synchrotron beamlines in order to make use of the appreciably larger f″ of sulfur at longer wavelengths in the range between 1.7 -2.5 Å [24][25][26][27][28][29][30] although Cu Kα is also feasible for sulfur SAD phasing [31][32][33]. Both copper and chromium anode wavelengths (1.54 and 2.29 Å) have been increasingly employed for the same purpose in lab X-ray sources with success [34][35][36].…”