Larkin Chris, Datta Saumen, Nezami Azin, Dohm Julie A, Schildbach Joel F
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
Acta Crystallogr D Biol Crystallogr. 2003 Aug;59(Pt 8):1514-6. doi: 10.1107/s0907444903012964. Epub 2003 Jul 23.
Conjugative plasmids are capable of transferring a copy of themselves in single-stranded form from donor to recipient bacteria. Prior to transfer, one plasmid strand must be cleaved in a sequence-specific manner by a relaxase or mobilization protein. TraI is the relaxase for the conjugative plasmid F factor. A 36 kDa N-terminal fragment of TraI possesses the single-stranded DNA-binding and cleavage activity of the protein. Crystals of the 36 kDa TraI fragment in native and selenomethionine-labeled forms were grown by sitting-drop vapor-diffusion methods using PEG 1000 as the precipitant. Crystallization in the presence of chloride salts of magnesium and strontium was required to obtain crystals yielding high-resolution diffraction. To maintain high-resolution diffraction upon freezing, crystals had to be soaked in crystallization buffer with stepwise increases of ethylene glycol. The resulting crystals were trigonal and diffracted to a resolution of 3.1 A or better using synchrotron radiation.
接合质粒能够以单链形式将自身的一个拷贝从供体细菌转移到受体细菌。在转移之前，一条质粒链必须被解旋酶或动员蛋白以序列特异性方式切割。TraI是接合质粒F因子的解旋酶。TraI的一个36 kDa N端片段具有该蛋白的单链DNA结合和切割活性。通过使用聚乙二醇1000作为沉淀剂的坐滴气相扩散法，培养了天然形式和硒代甲硫氨酸标记形式的36 kDa TraI片段的晶体。需要在镁和锶的氯化物盐存在下进行结晶，以获得能产生高分辨率衍射的晶体。为了在冷冻时保持高分辨率衍射，晶体必须浸泡在含有逐步增加的乙二醇的结晶缓冲液中。所得晶体为三角晶系，使用同步辐射可衍射至3.1 Å或更高的分辨率。
