The first step of phasing in any de novo protein structure determination using isomorphous replacement （IR） or anomalous scattering （AD） experiments is to find heavy atom positions. Traditionally, heavy atom positions can be solved by inspecting the difference Patterson maps. Due to the weak signals in isomorphous or anomalous differences and the noisy background in the Patterson map, the search for heavy atoms may become difficult. Here, the direct demodulation （DD） method is applied to the difference Patterson maps to reduce the noisy backgrounds and sharpen the signal peaks. The real space Patterson search by using these optimized maps can locate the heavy atom positions more accurately. It is anticipated that the direct demodulation method can assist in heavy atom position determination and facilitate the de novo structure determination of proteins.
MMP-12是癌症治疗药物靶标.为了更好研制新药,需要大量制备MMP-12,但MMP-12在大肠杆菌中以包涵体形式表达.因此如何优化蛋白复性过程是大量获取MMP-12蛋白的关键.采用核磁共振、稳态荧光法、外源性ANS(8-anilinol-naphthalenesulfonic acid)荧光探针三种方法监控MMP-12变性蛋白的再折叠过程,以探究其复性折叠机制.研究发现MMP-12再折叠中点值与对应的尿素浓度几乎相等(Cm≈4,mid-point of transition).不同尿素浓度中MMP-12的二维1H-15NHSQC(heteronuclear single quantum correlation)谱图显示,尿素浓度从4mol/L降低到3mol/L是MMP-12蛋白复性折叠的关键步骤.据此我们将MMP-12蛋白复性从常规的梯度透析复性方法改进成等容透析复性法(即确保尿素从4mol/L到3mol/L的浓度变化缓慢),实现复性收率提高一倍.
With the development of the XFEL （X-ray free electron laser）, high quality diffraction patterns from nanocrystals have been achieved. The nanocrystals with different sizes and random orientations are injected to the XFEL beams and the diffraction patterns can be obtained by the so-called ＂diffraction-and-destruction＂ mode. The recovery of orientations is one of the most critical steps in reconstructing the 3D structure of nanocrystals. There is already an approach to solve the orientation problem by using the automated indexing software in crystallography. However, this method cannot distinguish the twin orientations in the cases of the symmetries of Bravais lattices higher than the point groups. Here we propose a new method to solve this problem. The shape transforms of nanocrystals can be determined from all of the intensities around the diffraction spots, and then Fourier transformation of a single crystal cell is obtained. The actual orientations of the patterns can be solved by comparing the values of the Fourier transformations of the crystal cell on the intersections of all patterns. This so-called ＂multiple-common-line＂ method can distinguish the twin orientations in the XFEL diffraction patterns successfully.
核磁共振(nuclear magnetic resonance,NMR)是以原子核自旋的共振跃迁为探测对象的谱学方法.当自旋量子数不为零的原子核处于外磁场中时,会引起能级的Zeeman分裂.若再施加能量等于Zeeman能级差的射频场,则会诱发原子核自旋的共振跃迁,这种现象即为核磁共振.核自旋的共振频率与原子的类型有关,且受原子所处化学和物理环境的影响.此外,NMR能量较低,不会影响探测对象(常为分子)的状态.因此,NMR能够在无损条件下提供多种具有原子和分子分辨的物质组成、结构、形态、动态变化等丰富信息.
Coherent X-ray microscopy has advanced towards higher-energy, more brilliant sources over the past decade since its demonstrations, and many advancements have been made towards optimizing this imaging technique. Here we present both the experimental instrument for obtaining diffraction patterns and the primary reconstruction of yeast cell 2D projection. In addition, the characteristics of the existing optics at BL29XUL of SPring-8 Facility and the method of image reconstruction are discussed.
In this paper the solution conformation of the response regulator proteins from Deinococcus radiodurans was studied by small-angle X-ray scattering （SAXS）. The SAXS curves of Dr-rrA in solutions were obtained at Beamline 1W2A of Beijing Synchrotron Radiation Facility （BSRF）. Two possible conformations of the response regulator proteins, compact and incompact conformations, have been represented by the known crystallographic structures. And theoretical solution scattering curves of the two possible conformations were calculated and fitted to the experimental scattering curve of Dr-rrA, respectively. The result indicates that the solution conformation of the response regulator proteins is inclined to the compact one, which is in agreement with the result of biochemical experiments.
With high concentrations of hemoglobin （Hb） in red blood cells, self-interactions among these molecules could increase the propensities of their polymerization and aggregation. In the present work, high concentration Hb in solution and red blood cells were analyzed by small-angle X-ray scattering. Calculation of the effective structure factor indicates that the interaction of Hb molecules is the same when they are crowded together in both the cell and physiological saline. The Hb molecules stay individual without the formation of aggregates and clusters in cells.
