Enhancement of MAD/MIR phasing at low resolution and a new procedure for automatic phase extension

To achieve de novo protein structure determination of challenging cases, multi-wavelength anomalous diffraction(MAD) and multiple isomorphous replacement(MIR) phasing can be powerful tools to obtain low-resolution initial phases from heavy-atom derivative datasets, then phase extension is needed against high-resolution data to obtain accurate structures.In this context, we propose a direct-methods procedure here that could improve the initial low-resolution MAD/MIR phase quality.And accordingly, an automated process for extending initial phases to high resolution is also described.These two procedures are both implanted in the newly released IPCAS pipeline.Three cases are used to perform the test, including one set of 4.17 ? MAD data from a membrane protein and two sets of MAD/MIR data with derivatives truncated down to 6.80 ? and 6.90 ?, respectively.All the results have shown that the initial phases generated from the direct-methods procedure are better than that from the conventional MAD/MIR methods.The automated phase extensions for the latter two cases starting from 6.80 ? to 3.00 ? and 6.90 ? to 2.80 ? are proved to be successful, leading to complete models.This may provide convenient and reliable tools for phase improvement and phase extension in difficult low-resolution tasks.

SIR phasing by combination of SOLVE/RESOLVE and dual-space fragment extension involving OASIS

A new phasing procedure has been proposed for dealing with single isomorphous replacement （SIR） x-ray diffraction data. The procedure combines SOLVE/RESOLVE with the dual-space fragment extension involving OASIS. Two sets of SIR data at 0.28 nm resolution taken from the protein （R）-phycoerythrin （PDB code： 1LIA） were used in the test. For one of the two SIR data sets, a default run of SOLVE/RESOLVE based on the heavy-atom substructure found by SHLEXD led automatically to an interpretable electron density map. OASIS could not effectively improve the result. For the other set of SIR data, SOLVE/RESOLVE resulted in a fragmented model consisting of 454 of the total 668 residues, in which only 29 residues were docked into the sequence. Based on this model, 7 iteration cycles of OASIS-DM- RESOLVE （build only） yielded automatically a model of 547 residues with 133 residues docked into the sequence. The overall-averaged phase error decreased considerably and the quality of electron density map was improved significantly. Two more cycles of iterative OASIS-DM-RESOLVE were carried out, in which the output phases and figures of merit from DM were merged with that from the original run of SOLVE/RESOLVE before they were passed onto RESOLVE （build only）. This led automatically to a model containing 452 residues with 173 docked into the sequence. The resultant electron density map is manually traceable. It is concluded that when results of SOLVE/RESOLVE are not sufficiently satisfactory, the combination of SOLVE/RESOLVE and OASIS-DM-RESOLVE （build only） may significantly improve them.

Combining SAD/SIR iteration and MR iteration in partial-model extension of proteins

There are two kinds of dual-space partial-model extensions which involve the direct-method program OASIS. The first kind, named SAD/SIR iteration, uses SAD/SIR information, while the second kind, named molecular replacement （MR） iteration, does not use that information. In general, the SAD/SIR iteration is more powerful since more experimental information is used. However, in most cases when protein structures are solved with the molecular replacement method, SAD/SIR information is not available. Thus the MR iteration is particularly useful for the completion of models from molecular replacement. The SAD/SIR iteration will be automatically used in OASIS for data sets containing SAD/SIR signals, while the MR iteration will be dedicated to data sets without SAD/SIR signals. The present paper shows that for data containing SAD/SIR signals, a combination of SAD/SIR iteration and MR iteration could lead to significantly better results than that obtained from the SAD/SIR iteration alone.

New expression of bimodal phase distributions in direct-method phasing of protein single-wavelength anomalous diffraction data

One of the essential points of the direct-method single-wavelength anomalous diffraction （SAD） phasing for proteins is to express the bimodal SAD phase distribution by the sum of two Gaussian functions peaked respectively at φh″＋｜△φh｜ and φh″-｜△φh｜. The probability for △φh being positive （P＋） can be derived based on the Cochran distribution in direct methods. Hence the SAD phase ambiguity can be resolved by multiplying the Gaussian function peaked at φh″＋｜△φh｜ with P＋ and multiplying the Gaussian function peaked at φh″-｜△φh｜ with P_ （=1- P＋）. The direct-method SAD h phasing has been proved powerful in breaking SAD phase ambiguities, in particular when anomalous-scattering signals are weak. However, the approximation of bimodal phase distributions by the sum of two Gaussian functions introduces considerable errors. In this paper we show that a much better approximation can be achieved by replacing the two Gaussian functions with two von Mises distributions. Test results showed that this leads to significant improvement on the efficiency of direct-method SAD-phasing.

SAD phasing by OASIS at different resolutions down to 0.30 nm and below

Single-wavelength anomalous diffraction （SAD） phasing is increasingly important in solving de novo protein structures. Direct methods have been proved very efficient in SAD phasing. This paper aims at probing the low-resolution limit of direct-method SAD phasing. Two known proteins TT0570 and Tom70p were used as test samples. Sulfur-SAD data of the protein TT0570 were collected with conventional Cu-Kα source at 0.18 nm resolution. Its truncated subsets respectively at 0.21, 0.30, 0.35 and 0.40 nm resolutions were used in the test. TT0570 Cu-Kα sulfur-SAD data have an expected Bijvoet ratio 〈 ｜△F｜ 〉 / 〈 F 〉 ～ 0.55%. In the 0.21 nm case, a single run of OASIS-DM-ARP/wARP led automatically to a model containing 1178 of the total 1206 residues all docked into the sequence. In 0.30 and 0.35 nm cases, SAD phasing by OASIS-DM led to traceable electron density maps. In the 0.40 nm case, SAD phasing by OASIS-DM resulted in a degraded electron density map, which may be difficult to trace but still contains useful secondary-structure information. Test on real 0.33 nm selenium-SAD data of the protein Tom70p showed that even automatic model building was not successful, the combination of manual tracing and direct-method fragment extension was capable of significantly improving the electron-density map. This provides the possibility of effectively improving the manually built model before structure refinement is performed.

OASIS4.0—a new version of the program OASIS for phasing protein diffraction data

The program OASIS4.0 has been released. Apart from the improved single-wavelength anomalous diffraction （SAD） phasing algorithm described in a separate paper, an important new feature in this version is the automation of the iterative phasing and model-building process in solving protein structures. A new graphical user＇s interface （GUI） is provided for controlling and real-time monitoring the dual-space iterative process. The GUI is discussed in detail in the present paper.

直接法解超结构的改进

直接法用于求解晶体超结构，已证明是一种客观的、有效的方法．本文提出通过使各衍射组衍射数目相等或Ｅ值均衡指派，均可收到提高直接法解超结构能力的效果．

直接法应用于蛋白质二维晶体的电子晶体学图像处理

试把直接法应用于抗生蛋白链菌素 (streptavidin)沿 [0 0 1]方向投影的模拟像和相应的理论结构因子作图像处理 .先用两张高分辨电子显微像作直接法解卷 ,以互补因衬度传递函数的作用而损失的结构信息 ,求得的欠焦值比用单张像解卷的结果更接近真实值 .把从结构模型计算出的 0 30nm以内的相位 ,以及 0 2 5nm以内的振幅作为起始数据 ,进行直接法相位外推 ,并借助团簇分析方法得到 0 30nm至 0 2 5nm之间的相位 .所得分辨率为 0 30nm的解卷像和分辨率为 0 2 5nm的晶体结构投影均与理论结构模型的相应投影电势图一致 .