Srivastava Vijay Kumar, Rana Ajay Kumar, Sahasrabuddhe Amogh A, Gupta C M, Pratap J V
Molecular and Structural Biology Division, CSIR - Central Drug Research Institute, B.S/10/1, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow 226 021, India.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 May 1;69(Pt 5):535-9. doi: 10.1107/S1744309113007811. Epub 2013 Apr 30.
Leishmania donovani coronin CRN12 is an actin-binding protein which consists of two domains: an N-terminal WD repeat domain and a C-terminal coiled-coil domain. The coiled-coil domain is 53 residues in length. Helix-helix interactions in general and coiled coils in particular are ubiquitous in the structure of proteins and play a significant role in the association among proteins, including supramolecular assemblies and transmembrane receptors that mediate cellular signalling, transport and actin dynamics. The L. donovani coronin CRN12 coiled-coil domain (5.8 kDa) was cloned, overexpressed, purified to homogeneity and the N-terminal 6×His tag was successfully removed by thrombin cleavage. Crystals of recombinant L. donovani coronin CRN12 coiled-coil domain were grown by vapour diffusion using a hanging-drop setup. Diffraction-quality crystals were obtained and data extending to 2.46 Å resolution were collected at 100 K on BM14, ESRF, Grenoble, France. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 118.0, b = 50.6, c = 46.0 Å, β = 111.0°. Matthews coefficient (VM) calculations suggested the presence of 4-6 molecules in the asymmetric unit, corresponding to a solvent content of ∼33-55%, and are consistent with self-rotation function calculations.
杜氏利什曼原虫冠蛋白CRN12是一种肌动蛋白结合蛋白，由两个结构域组成：一个N端WD重复结构域和一个C端卷曲螺旋结构域。卷曲螺旋结构域长度为53个残基。一般来说，螺旋-螺旋相互作用，尤其是卷曲螺旋，在蛋白质结构中普遍存在，并且在蛋白质之间的缔合中发挥重要作用，包括介导细胞信号传导、运输和肌动蛋白动力学的超分子组装体和跨膜受体。克隆、过表达并纯化了杜氏利什曼原虫冠蛋白CRN12卷曲螺旋结构域（5.8 kDa），使其达到均一性，并通过凝血酶切割成功去除了N端的6×His标签。使用悬滴装置通过气相扩散法培养重组杜氏利什曼原虫冠蛋白CRN12卷曲螺旋结构域的晶体。获得了衍射质量良好的晶体，并在法国格勒诺布尔欧洲同步辐射装置（ESRF）的BM14上于100 K收集了分辨率高达2.46 Å的数据。该晶体属于单斜空间群C2，晶胞参数为a = 118.0、b = 50.6、c = 46.0 Å，β = 111.0°。马修斯系数（VM）计算表明不对称单位中存在4 - 6个分子，对应溶剂含量约为33 - 55%，这与自旋转函数计算结果一致。
