The refinement of a molecular model is a computational procedure by which the atomic model is fitted to the diffraction data. The commonly used target in the refinement of macromolecular structures is the maximum-likelihood (ML) function, which relies on the assessment of model errors. The current ML functions rely on cross-validation. They utilize phase-error estimates that are calculated from a small fraction of diffraction data, called the test set, that are not used to fit the model. An approach has been developed that uses the work set to calculate the phase-error estimates in the ML refinement from simulating the model errors via the random displacement of atomic coordinates. It is called ML free-kick refinement as it uses the ML formulation of the target function and is based on the idea of freeing the model from the model bias imposed by the chemical energy restraints used in refinement. This approach for the calculation of error estimates is superior to the cross-validation approach: it reduces the phase error and increases the accuracy of molecular models, is more robust, provides clearer maps and may use a smaller portion of data for the test set for the calculation of Rfree or may leave it out completely
COBISS.SI-ID: 1537009860
At present, the determination of crystal structures from data that have been acquired from twinned crystals is routine; however, with the increase of crystal structures, additional crystal lattice disorders are discovered. Here, we present a previously undescribed partial rotational order-disorder that has been observed in the crystals of stefin B. The diffraction images reveal normal diffraction patterns that result from a regular crystal lattice. The data could be processed in I4 and I422 space groups, yet one crystal exhibited a notable rejection rate in the higher symmetry space group. We found an explanation for this behaviour once the crystal structures were solved and refined and electron density maps were inspected. The lattice of stefin B crystals is composed of five tetramer layers: four well-ordered layers, which are followed by an additional layer of alternatively placed tetramers. The presence of alternative positions was revealed by the inspection of score maps of electron density. The well-ordered layers correspond to the crystal symmetry of the I422 space group. In addition, the positions of molecules in the additional layer are related by twofold rotational axes, which correspond to the I422 space group; however, these molecules lie on the twofold axis and can be related only in a statistical manner. When occupancies of alternate positions and overlapping are equal, the crystal lattice indeed fulfills the criteria of the I422 space group; when these occupancies are not equal, the lattice fulfills the criteria of the I4 space group only.
COBISS.SI-ID: 27622183
The recently identified fungal protease inhibitors cnispin, from Clitocybe nebularis, and cospin, from Coprinopsis cinerea, are both β-trefoil proteins highly specific for trypsin. The reactive site residue of cospin, Arg27, is located on the β2-β3 loop. We show here, that the reactive site residue in cnispin is Lys127, located on the β11-β12 loop. Cnispin is a substrate-like inhibitor and the β11-β12 loop is yet another β-trefoil fold loop recruited for serine protease inhibition. By site-directed mutagenesis of the P1 residues in the β2-β3 and the β11-β12 loops in cospin and cnispin, protease inhibitors with different specificities for trypsin and chymotrypsin inhibition have been engineered. Double headed inhibitors of trypsin or trypsin and chymotrypsin were prepared by introducing a second specific site residue into the β2-β3 loop in cnispin and into the β11-β12 loop in cospin. These results show that β-trefoil protease inhibitors from mushrooms exhibit broad plasticity of loop utilization in protease inhibition.
COBISS.SI-ID: 27828775
Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.
COBISS.SI-ID: 27410471