Search Results (3)

Temperature directly influences the function and structure of proteins. Crystal structures determined at room temperature offer more biologically relevant structural information regarding flexibility, rigidity, and thermal motion than those determined by conventional cryocrystallography. Crystal structures can be determined at room temperature using conventional macromolecular crystallography (MX) or serial crystallography (SX) techniques. Among these, MX may theoretically be affected by radiation damage or X-ray heating, potentially resulting in differences between the room temperature structures determined by MX and SX, but this has not been fully elucidated. In this study, the room temperature structure of xylanase GH11 from Thermoanaerobacterium saccharolyticum was determined by MX (RT-TsaGH11-MX). The RT-TsaGH11-MX exhibited both the open and closed conformations of the substrate-binding cleft within the β-sandwich fold. The RT-TsaGH11-MX showed distinct structural changes and molecular flexibility when compared with the RT-TsaGH11 determined via serial synchrotron crystallography. The notable molecular conformation and flexibility of the RT-TsaGH11-MX may be induced by radiation damage and X-ray heating. These findings will broaden our understanding of the potential limitations of room temperature structures determined by MX. Full article

Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions. Although X-ray crystallography has been the method of choice for solving macromolecular structures for many years, recently electron microscopy has also become an increasingly powerful structure-solving technique. We have used our previous experience with cryo-crystallography to develop a method to prepare cryo-EM grids in an anaerobic chamber and have applied it to solve the structures of apoferritin and the 3 [Fe₄S₄]-containing pyruvate ferredoxin oxidoreductase (PFOR) at 2.40 Å and 2.90 Å resolution, respectively. The maps are of similar quality to the ones obtained under air, thereby validating our method as an improvement in the structural investigation of oxygen-sensitive metalloproteins by cryo-EM. Full article

Cryocrystallography is a widely used method for determining the crystal structure of macromolecules. This technique uses a cryoenvironment, which significantly reduces the radiation damage to the crystals and has the advantage of requiring only one crystal for structural determination. In standard cryocrystallography, a single crystal is used for collecting diffraction data, which include single-crystal diffraction patterns. However, the X-ray data recorded often may contain diffraction patterns from several crystals. The indexing of multicrystal diffraction patterns in cryocrystallography requires more precise data processing techniques and is therefore time consuming. Here, an approach for processing multicrystal diffraction data using a serial crystallography program is introduced that allows for the integration of multicrystal diffraction patterns from a single image. Multicrystal diffraction data were collected from lysozyme crystals and processed using the serial crystallography program CrystFEL. From 360 images containing multicrystal diffraction patterns, 1138 and 691 crystal lattices could be obtained using the XGANDALF and MOSFLM indexing algorithms, respectively. Using this indexed multi-lattice information, the crystal structure of the lysozyme could be determined successfully at a resolution of 1.9 Å. Therefore, the proposed approach, which is based on serial crystallography, is suitable for processing multicrystal diffraction data in cryocrystallography. Full article