Ockwell D M, Hough M A, Grossmann J G, Hasnain S S, Hao Q
Department of Chemistry, De Montfort University, Leicester LE1 9BH, England.
Acta Crystallogr D Biol Crystallogr. 2000 Aug;56(Pt 8):1002-6. doi: 10.1107/s0907444900007721.
Solution of the phase problem is central to crystallographic structure determination. The conventional methods of isomorphous replacement (MIR or SIR) and molecular replacement are ineffective in the absence of a suitable isomorphous heavy-atom derivative or knowledge of the structure of a homologous protein. A recent method utilizing the low-resolution molecular shape determined from solution X-ray scattering data has shown to be successful in locating the molecular shape within the crystallographic unit cell in the case of the trimer nitrite reductase (NiR, 105 kDa) [Hao et al. (1999), Acta Cryst. D55, 243-246]. This was achieved by performing a direct real-space search for orientation and translation using knowledge of the orientation of the polar angles of the non-crystallographic axis obtained by performing a self-rotation on crystallographic data. This effectively reduces the potential six-dimensional search to a four-dimensional one (Eulerian angle gamma and three translational parameters). In the case of NiR, the direct four-dimensional search produced a clear solution that was in good agreement with the known structure. The program FSEARCH incorporating this method has been generalized to handle molecules from all space groups and in particular those in possession of non-crystallographic symmetry. However, the method employed was initially unsuccessful when applied to the small dimeric molecule superoxide dismutase (SOD, 32 kDa) owing to the absence of strong reflections at low resolution caused by saturation at the detector. The determined solution deviated greatly from that of the known structure [Hough & Hasnain (1999), J. Mol. Biol. 287, 579-592]. It was found that once these absent reflections were replaced by a series of randomly generated intensity values and cluster analysis was performed on the output, the signal-to-noise ratio was improved and a most probable solution was found. The electron-density map of the stochastically determined solution agrees well with the known structure; the phase error calculated from this map was 67 degrees within 14 A resolution.
相位问题的解决对于晶体结构测定至关重要。在没有合适的同晶型重原子衍生物或同源蛋白质结构信息的情况下，传统的同晶型置换法（MIR或SIR）和分子置换法均无效。最近一种利用从溶液X射线散射数据确定的低分辨率分子形状的方法，已证明在三聚体亚硝酸还原酶（NiR，105 kDa）的情况下，成功地在晶体学晶胞内定位了分子形状[Hao等人（1999年），《晶体学报》D55卷，243 - 246页]。这是通过利用对晶体学数据进行自旋转获得的非晶体学轴极角方向的知识，在实空间中直接搜索方向和平移来实现的。这有效地将潜在的六维搜索减少到了四维搜索（欧拉角γ和三个平移参数）。对于NiR，直接的四维搜索产生了一个与已知结构高度吻合的清晰解。包含此方法的程序FSEARCH已被推广，以处理所有空间群的分子，特别是那些具有非晶体学对称性的分子。然而，该方法最初应用于小的二聚体分子超氧化物歧化酶（SOD，32 kDa）时并不成功，这是由于探测器饱和导致低分辨率下缺乏强反射。所确定的解与已知结构有很大偏差[Hough & Hasnain（1999年），《分子生物学杂志》287卷，579 - 592页]。结果发现，一旦用一系列随机生成的强度值替换这些缺失的反射，并对输出进行聚类分析，信噪比就会提高，并找到了最可能的解。随机确定的解的电子密度图与已知结构吻合良好；从该图计算出的相位误差在14 Å分辨率内为67度。
