This study focuses on the effects of the organic ligand 4-ethylresorcinol on the crystal structure of human insulin using powder X-ray crystallography. For this purpose, systematic crystallization experiments have been conducted in the presence of the organic ligand and zinc ions within the pH range 4.50-8.20, while observing crystallization behaviour around the isoelectric point of insulin. Highthroughput crystal screening was performed using a laboratory X-ray diffraction system. The most representative samples were selected for synchrotron X-ray diffraction measurements, which took place at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS). Four different crystalline polymorphs have been identified. Among these, two new phases with monoclinic symmetry have been found, which are targets for the future development of microcrystalline insulin drugs.

The primary focus of the present work is the study of the effects that two ligands and the crystallization pH have on the crystalline forms of human insulin. For this purpose, human insulin (HI) was co-crystallized with two distinct phenolic derivatives: the organic ligands meta-cresol (m-cresol) and 4-nitrophenol. The formation of polycrystalline precipitates was then followed by means of structural characterization of the individual specimens in terms of unit-cell symmetry and parameters. In both cases, two different polymorphs were identified via X-ray powder diffraction measurements, the first of hexagonal symmetry (R3 space group) at higher pH values and the second of monoclinic symmetry (space group P21) with unit-cell parameters a = 87.4282 (5), b = 70.5020 (3), c = 48.3180 (4) Å, β = 106.8958 (4)°, the latter of which to our knowledge has never been observed before.

Natural or artificially manufactured peptides attract scientific interest worldwide owing to their wide array of pharmaceutical and biological activities. X-ray structural studies are used to provide a precise extraction of information, which can be used to enable a better understanding of the function and physicochemical characteristics of peptides. Although it is vulnerable to disassociation, one of the most vital human peptide hormones, somatostatin, plays a regulatory role in the endocrine system as well as in the release of numerous secondary hormones. This study reports the successful crystallization and complete structural model of octreotide, a stable octapeptide analogue of somatostatin. Common obstacles in crystallographic studies arising from the intrinsic difficulties of obtaining a suitable single-crystal specimen were efficiently overcome as polycrystalline material was employed for synchrotron and laboratory X-ray powder diffraction (XPD) measurements. Data collection and preliminary analysis led to the identification of unit-cell symmetry [orthorhombic, P2 1 2 1 2 1 , a = 18.5453 (15), b = 30.1766 (25), c = 39.798 (4) Å ], a process which was later followed by complete structure characterization and refinement, underlying the efficacy of the suggested (XPD) approach.

Abstract.Following the seminal work of Von Dreele, high quality powder X-ray diffraction studies on proteins are being established as a valuable complementary technique to singlecrystal measurements. Several studies using a variety of experiments approaches have been reported in the literature, including high-resolution studies employing parallel beam geometry and high intensity measurements using position sensitive detectors. The choice of the optimum instrumental configuration depends on a number of competing factors such as the amount of sample available, its radiation sensitivity, and the quality of the data required for data analysis, e.g. angular resolution, the extent of the data in d-spacing, or the number of patterns required to explore the protein's behaviour at different temperatures, or under different crystallisation conditions, etc. Here we discuss several advantages and disadvantages of different data collection methods followed for selected examples of small proteins.

The effect of relative humidity (rH) on protein crystal structures, an area that has attracted high scientific interest during the past decade, is investigated in this study on hen egg‐white lysozyme (HEWL) polycrystalline precipitates via in situ laboratory X‐ray powder diffraction (XRPD) measurements. For this purpose, HEWL was crystallized at room temperature and pH 4.5, leading to a novel monoclinic HEWL phase which, to our knowledge, has not been reported before. Analysis of XRPD data collected upon rH variation revealed several structural modifications. These observations, on a well‐studied molecule like HEWL, underline not only the high impact of humidity levels on biological crystal structures, but also the significance of in‐house XRPD as an analytical tool in industrial drug development and its potential to provide information for enhancing manufacturing of pharmaceuticals.

Variation of relative humidity (rH) greatly affects the internal order of solventbased protein crystals, and the rearrangement of molecules can be efficiently recorded in distinct diffraction patterns. This study focuses on this topic, reporting the effect of rH variation experiments on hen egg white lysozyme (HEWL) polycrystalline precipitates of tetragonal symmetry using X-ray powder diffraction (XRPD). In situ XRPD data were collected on HEWL specimens during dehydration and rehydration processes using laboratory instrumentation. A known polymorph [space group P4 3 2 1 2, a = 79.07181 (1), c = 38.0776 (1) Å ] was identified during gradual dehydration from 95 to 63% rH and vice versa. Pawley analysis of collected data sets and accurate extraction of unitcell parameters indicated a characteristic evolution of the tetragonal axes with rH. In addition, there is a low humidity level below which samples do not retain their crystallinity. This work illustrates the accuracy of laboratory XRPD as a probe for time-resolved studies of proteins and in situ investigations of gradual structural modifications upon rH variation. These experiments provide essential information for improving production and post-production practices of microcrystalline protein-based pharmaceuticals. research papers J. Appl. Cryst. (2019). 52, 816-827 S. Logotheti et al. Effects of relative humidity on HEWL 817

Viral proteases are proteolytic enzymes that orchestrate the assembly of viral components during the viral life cycle and proliferation. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis are presented of protease 3C, the main protease of an emerging enterovirus, coxsackievirus B3, that is responsible for many cases of viral myocarditis. Polycrystalline protein precipitates suitable for X-ray powder diffraction (XRPD) measurements were produced in the presence of 22-28%(w/v) PEG 4000, 0.1 M Tris-HCl, 0.2 M MgCl in a pH range from 7.0 to 8.5. A polymorph of monoclinic symmetry (space group C2, unit-cell parameters a = 77.9, b = 65.7, c = 40.6 Å, β = 115.9°) was identified via XRPD. These results are the first step towards the complete structural determination of the molecule via XRPD and a parallel demonstration of the accuracy of the method.

Flavivirus infections often provoke life-threatening diseases of epidemic magnitudes, thus extensive research is currently directed towards the development of efficient vaccines and approved antiviral compounds. We present here the expression, purification, crystallization and preliminary X-ray diffraction analysis of one of the components of the flavivirus replication complex, the non-structural protein 5 (NS5) mRNA methyltransferase (MTase) domain, from an emerging pathogenic flavivirus, dengue virus 3 (DEN3). Polycrystalline precipitates of DEN3 NS5 MTase, suitable for X-ray powder diffraction (XRPD) measurements, were produced in the presence of PEG 8000 (25–32.5% (w/v)), 0.1 M Tris-Amino, in a pH range from 7.0 to 8.0. A polymorph of orthorhombic symmetry (space group: