The CCP4 suite : integrative software for macromolecular crystallography

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 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

Chitinase Chit62J4 Essential for Chitin Processing by Human Microbiome Bacterium Clostridium paraputrificum J4

Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N′-diacetyl-β-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with Km = 0.24 mM and kcat = 30.0 s−1. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains—2x Fn3/Big3 and a carbohydrate binding module—that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria

Atomic resolution studies of S1 nuclease complexes reveal details of RNA interaction with the enzyme despite multiple lattice-translocation defects

S1 nuclease from Aspergillus oryzae is a single-strand-specific nuclease from the S1/P1 family that is utilized in biochemistry and biotechnology. S1 nuclease is active on both RNA and DNA but with differing catalytic efficiencies. This study clarifies its catalytic properties using a thorough comparison of differences in the binding of RNA and DNA in the active site of S1 nuclease based on X-ray structures, including two newly solved complexes of S1 nuclease with the products of RNA cleavage at atomic resolution. Conclusions derived from this comparison are valid for the whole S1/P1 nuclease family. For proper model building and refinement, multiple lattice-translocation defects present in the measured diffraction data needed to be solved. Two different approaches were tested and compared. Correction of the measured intensities proved to be superior to the use of the dislocation model of asymmetric units with partial occupancy of individual chains. As the crystals suffered from multiple lattice translocations, equations for their correction were derived de novo. The presented approach to the correction of multiple lattice-translocation defects may help to solve similar problems in the field of protein X-ray crystallography

Crystallographic fragment screening-based study of a novel FAD-dependent oxidoreductase from Chaetomium thermophilum

The FAD-dependent oxidoreductase from Chaetomium thermophilum (CtFDO) is a novel thermostable glycoprotein from the glucose–methanol–choline (GMC) oxidoreductase superfamily. However, CtFDO shows no activity toward the typical substrates of the family and high-throughput screening with around 1000 compounds did not yield any strongly reacting substrate. Therefore, protein crystallography, including crystallographic fragment screening, with 42 fragments and 37 other compounds was used to describe the ligand-binding sites of CtFDO and to characterize the nature of its substrate. The structure of CtFDO reveals an unusually wide-open solvent-accessible active-site pocket with a unique His–Ser amino-acid pair putatively involved in enzyme catalysis. A series of six crystal structures of CtFDO complexes revealed five different subsites for the binding of aryl moieties inside the active-site pocket and conformational flexibility of the interacting amino acids when adapting to a particular ligand. The protein is capable of binding complex polyaromatic substrates of molecular weight greater than 500 Da

Protein Binder (ProBi) as a New Class of Structurally Robust Non-Antibody Protein Scaffold for Directed Evolution

Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity

Structural and Catalytic Properties of S1 Nuclease from Aspergillus oryzae Responsible for Substrate Recognition, Cleavage, Non–Specificity, and Inhibition

The single–strand–specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1–P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X–ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1–P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes–shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1–P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity