The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The... Show moreThe Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world. Show less
Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to... Show moreStreptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P-1-type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Delta ccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance. Show less
GlxA from Streptomyces lividans is a mononuclear copper-radical oxidase and a member of the auxiliary activity family 5 (AA5). Its domain organisation and low sequence homology make it a distinct... Show moreGlxA from Streptomyces lividans is a mononuclear copper-radical oxidase and a member of the auxiliary activity family 5 (AA5). Its domain organisation and low sequence homology make it a distinct member of the AA5 family in which the fungal galactose 6-oxidase (Gox) is the best characterised. GlxA is a key cuproenzyme in the copper-dependent morphological development of S. lividans with a function that is linked to the processing of an extracytoplasmic glycan. The catalytic sites in GlxA and Gox contain two distinct one-electron acceptors comprising the copper ion and a 3′-(S-cysteinyl) tyrosine. The latter is formed post-translationally through a covalent bond between a cysteine and a copper-co-ordinating tyrosine ligand and houses a radical. In GlxA and Gox, a second co-ordination sphere tryptophan residue (Trp288 in GlxA) is present, but the orientation of the indole ring differs between the two enzymes, creating a marked difference in the π–π stacking interaction of the benzyl ring with the 3′-(S-cysteinyl) tyrosine. Differences in the spectroscopic and enzymatic activity have been reported between GlxA and Gox with the indole orientation suggested as a reason. Here, we report a series of in vivo and in vitro studies using the W288F and W288A variants of GlxA to assess the role of Trp288 on the morphology, maturation, spectroscopic and enzymatic properties. Our findings point towards a salient role for Trp288 in the kinetics of copper loading and maturation of GlxA, with its presence essential for stabilising the metalloradical site required for coupling catalytic activity and morphological development. Show less
