Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behaviour (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallographic community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.

Coot is a tool widely used for model building, refinement, and validation of macromolecular structures. It has been extensively used for crystallography and, more recently, improvements have been introduced to aid in cryo‐EM model building and refinement, as cryo‐EM structures with resolution ranging 2.5–4 A are now routinely available. Model building into these maps can be time‐consuming and requires experience in both biochemistry and building into low‐resolution maps. To simplify and expedite the model building task, and minimize the needed expertise, new tools are being added in Coot. Some examples include morphing, Geman‐McClure restraints, full‐chain refinement, and Fourier‐model based residue‐type‐specific Ramachandran restraints. Here, we present the current state‐of‐the‐art in Coot usage.

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In eukaryotes, dihydropyrimidinase catalyzes the second step of the reductive pyrimidine degradation, the reversible hydrolytic ring opening of dihydropyrimidines. Here we describe the three-dimensional structures of dihydropyrimidinase from two eukaryotes, the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum, determined and refined to 2.4 and 2.05 Å , respectively. Both enzymes have a (␤/␣) 8 -barrel structural core embedding the catalytic di-zinc center, which is accompanied by a smaller ␤-sandwich domain. Despite loop-forming insertions in the sequence of the yeast enzyme, the overall structures and architectures of the active sites of the dihydropyrimidinases are strikingly similar to each other, as well as to those of hydantoinases, dihydroorotases, and other members of the amidohydrolase superfamily of enzymes. However, formation of the physiologically relevant tetramer shows subtle but nonetheless significant differences. The extension of one of the sheets of the ␤-sandwich domain across a subunit-subunit interface in yeast dihydropyrimidinase underlines its closer evolutionary relationship to hydantoinases, whereas the slime mold enzyme shows higher similarity to the noncatalytic collapsin-response mediator proteins involved in neuron development. Catalysis is expected to follow a dihydroorotase-like mechanism but in the opposite direction and with a different substrate. Complexes with dihydrouracil and N-carbamyl-␤-alanine obtained for the yeast dihydropyrimidinase reveal the mode of substrate and product binding and allow conclusions about what determines substrate specificity, stereoselectivity, and the reaction direction among cyclic amidohydrolases.Dihydropyrimidinase (DHPase, 2 dihydropyrimidine amidohydrolase, EC 3.5.2.2) catalyzes the ring opening of 5,6-dihydrouracil to N-carbamyl-␤-alanine and of 5,6-dihydrothymine to N-carbamyl-␤-amino isobutyrate, which represents the second step in the three-step reductive degradation pathway of uracil, thymine, and several anti-cancer drugs (1, 2). The ring cleavage reaction is reversible and is achieved by hydrolysis of the amide bond between nitrogen 3 and carbon 4 of the dihydropyrimidine ring (Scheme 1).As an integral part of the pyrimidine catabolic pathway, DHPase activity is required for the regulation of the pyrimidine pool size available for nucleic acid synthesis and for supplying the cell with ␤-alanine. The reductive degradation of uracil represents the major pathway providing the non-proteinogenic amino acid in plants and filamentous fungi and its sole source in mammalian tissues (3). It is thought to have neurotransmitter function because of its chemical similarity to ␥-amino-n-butyric acid and glycine (4), and it is accumulated in biologically active dipeptides such as carnosin and anserine.The medical condition of DHPase deficiency (dihydropyrimidinuria) is a rare event and has thus far been described for five patients who showed a variable clinical phenotype consisting of seizures, mental retardation, growth retardat...