Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74-motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme-product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c-di-GMP in crystal resulted in a distinguished complex with the emerging product 5'-GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies.

• Keywords
• Stenotrophomonas maltophilia, RNA, S1/P1 nuclease, X‐ray crystallography, c‐di‐GMP cleavage,
• MeSH
• Bacterial Proteins metabolism   chemistry   genetics   MeSH
• Cyclic GMP metabolism   analogs & derivatives   chemistry   MeSH
• Catalytic Domain * MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• RNA metabolism   chemistry   genetics   MeSH
• Stenotrophomonas maltophilia * enzymology   genetics   metabolism   MeSH
• Substrate Specificity MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Cyclic GMP MeSH
• RNA MeSH

Binder H33 is a small protein binder engineered by ribosome display to bind human interleukin 10. Crystals of binder H33 display severe diffraction anisotropy. A set of data files with correction for diffraction anisotropy based on different local signal-to-noise ratios was prepared. Paired refinement was used to find the optimal anisotropic high-resolution diffraction limit of the data: 3.13-2.47 Å. The structure of binder H33 belongs to the 2% of crystal structures with the highest solvent content in the Protein Data Bank.

• Keywords
• anisotropy, binder H33, paired refinement,
• Publication type
• Journal Article MeSH

The resistance of the emerging human pathogen Stenotrophomonas maltophilia to tetracycline antibiotics mainly depends on multidrug efflux pumps and ribosomal protection enzymes. However, the genomes of several strains of this Gram-negative bacterium code for a FAD-dependent monooxygenase (SmTetX) homologous to tetracycline destructases. This protein was recombinantly produced and its structure and function were investigated. Activity assays using SmTetX showed its ability to modify oxytetracycline with a catalytic rate comparable to those of other destructases. SmTetX shares its fold with the tetracycline destructase TetX from Bacteroides thetaiotaomicron; however, its active site possesses an aromatic region that is unique in this enzyme family. A docking study confirmed tetracycline and its analogues to be the preferred binders amongst various classes of antibiotics.

• Keywords
• FAD-dependent monooxygenases, antibiotic resistance, tetracycline,
• MeSH
• Anti-Bacterial Agents pharmacology   chemistry   MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Oxytetracycline * metabolism   MeSH
• Stenotrophomonas maltophilia * genetics   metabolism   MeSH
• Tetracycline pharmacology   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Oxytetracycline * MeSH
• Tetracycline MeSH

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.

• Keywords
• CCP4, Collaborative Computational Project No. 4, crystallography software, macromolecular crystallography,
• MeSH
• Crystallography, X-Ray MeSH
• Macromolecular Substances MeSH
• Proteins * chemistry   MeSH
• Software * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Macromolecular Substances MeSH
• Proteins * MeSH

In macromolecular crystallography, paired refinement is generally accepted to be the optimal approach for the determination of the high-resolution cutoff. The software tool PAIREF provides automation of the protocol and associated analysis. Support for phenix.refine as a refinement engine has recently been implemented in the program. This feature is presented here using previously published data for thermolysin. The results demonstrate the importance of the complete cross-validation procedure to obtain a thorough and unbiased insight into the quality of high-resolution data.

• Keywords
• PAIREF, Phenix, X-ray diffraction, high-resolution limit, macromolecular crystallography, paired refinement,
• MeSH
• Databases, Protein * standards   MeSH
• Crystallography, X-Ray methods   standards   MeSH
• Software * standards   MeSH
• Publication type
• Journal Article MeSH