The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae.
The difficult case of crystallization and structure solution for the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae.
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DOI：
10.1371/journal.pone.0003201
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发表时间：
2008-09-12
期刊：
影响因子：
3.7
通讯作者：
Sanderson MR
中科院分区：
文献类型：
--
作者：
Sohi MK;Veselkov DA;Laponogov I;Pan XS;Fisher LM;Sanderson MR
肺炎链球菌是社区获得性肺炎的主要原因，也与支气管炎、脑膜炎、耳炎和鼻窦炎有关。对青霉素和其他抗生素耐药性的出现和日益普遍，引起了人们对其他抗肺炎球菌药物的兴趣，例如靶向DNA促旋酶和拓扑异构酶IV的喹诺酮类药物。在结晶过程中，并在寻找一种方法，以确定阶段的拓扑异构酶IV从肺炎链球菌的ParC 55断裂-团聚结构域的结构，在该过程的每个阶段面临的障碍。这些问题包括：大多数晶体是孪生的，或者是无衍射的，或者是表现出高的镶嵌扩展。在有利于衍射的条件下生长的晶体很难在不失去衍射的情况下快速冷冻。由于问题的复杂性，通过分子置换的初始结构解决方案失败了，并且该方法被证明是不可行的。最终，成功的结构解决方案需要深入的数据分析和非常详细的分子置换搜索。对晶体抗孪晶剂进行了试验，并对两种不同的速冻方法进行了比较。晶体的脆弱性不允许将晶体转移到重原子溶液中的通常方法。因此，有必要在重原子化合物的存在下共结晶。多个同晶型置换方法是不成功的，因为工程化的7个半胱氨酸突变体不能成功地衍生化。最终，通过使用CNS对空间群P1中的大量溶液进行排序，使用分子替换来解决结构问题。本文的主要目的是描述所面临的障碍和克服，以获得数据集，这种困难的晶体，并确定相成功的结构解决方案。
Streptococcus pneumoniae is the major cause of community-acquired pneumonia and is also associated with bronchitis, meningitis, otitis and sinusitis. The emergence and increasing prevalence of resistance to penicillin and other antibiotics has led to interest in other anti-pneumonococcal drugs such as quinolones that target the enzymes DNA gyrase and topoisomerase IV. During crystallization and in the avenues to finding a method to determine phases for the structure of the ParC55 breakage-reunion domain of topoisomerase IV from Streptococcus pneumoniae, obstacles were faced at each stage of the process. These problems included: majority of the crystals being twinned, either non-diffracting or exhibiting a high mosaic spread. The crystals, which were grown under conditions that favoured diffraction, were difficult to flash-freeze without loosing diffraction. The initial structure solution by molecular replacement failed and the approach proved to be unviable due to the complexity of the problem. In the end the successful structure solution required an in-depth data analysis and a very detailed molecular replacement search. Crystal anti-twinning agents have been tested and two different methods of flash freezing have been compared. The fragility of the crystals did not allow the usual method of transferring the crystals into the heavy atom solution. Consequently, it was necessary to co-crystallize in the presence of the heavy atom compound. The multiple isomorphous replacement approach was unsuccessful because the 7 cysteine mutants which were engineered could not be successfully derivatized. Ultimately, molecular replacement was used to solve the structure by sorting through a large number of solutions in space group P1 using CNS. The main objective of this paper is to describe the obstacles which were faced and overcome in order to acquire data sets on such difficult crystals and determine phases for successful structure solution.
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肺炎链球菌的断裂 - 重新结构域IV：革兰氏阳性喹诺酮靶标的晶体结构。
影响因子：
3.7
作者：
Laponogov I;Veselkov DA;Sohi MK;Pan XS;Achari A;Yang C;Ferrara JD;Fisher LM;Sanderson MR
通讯作者：
Sanderson MR
影响因子：
4.9
作者：
Pan, XS;Fisher, LM
通讯作者：
Fisher, LM
影响因子：
64.8
作者：
STOCK, AM;MOTTONEN, JM;SCHUTT, CE
通讯作者：
SCHUTT, CE
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL
影响因子：
4.8
作者：
Leo, E;Gould, KA;Fisher, LM
通讯作者：
Fisher, LM