title: Cloning, overexpression, purification and crystallization of the CRN12 coiled-coil domain from Leishmania donovani.
abstract: 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.
authors.affiliation: Molecular and Structural Biology Division, CSIR - Central Drug Research Institute, B.S/10/1, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow 226 021, India.
authors.forename: Vijay Kumar
authors.initials: VK
authors.lastname: Srivastava
authors.forename: Ajay Kumar
authors.initials: AK
authors.lastname: Rana
authors.forename: Amogh A
authors.initials: AA
authors.lastname: Sahasrabuddhe
authors.forename: C M
authors.initials: CM
authors.lastname: Gupta
authors.forename: J V
authors.initials: JV
authors.lastname: Pratap
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003460:Crystallization; D005786:Gene Expression Regulation; D007893:Leishmania donovani / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D015800:Protozoan Proteins
keywords: CRN12 coiled-coil domain; Leishmania donovani