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The polymorphism of human insulin upon pH variation was characterized via X‐ray powder diffraction, employing a crystallization protocol previously established for co‐crystallization with phenolic derivatives. Two distinct rhombohedral (R3) polymorphs and one cubic (I213) polymorph were identified with increasing pH, corresponding to the T6, T3R3f and T2 conformations of insulin, respectively. The structure of the cubic T2 polymorph was determined via multi‐profile stereochemically restrained Rietveld refinement at 2.7 Å resolution. This constitutes the first cubic insulin structure to be determined from crystals grown in the presence of zinc ions, although no zinc binding was observed. The differences of the polycrystalline variant from other cubic insulin structures, as well as the nature of the pH‐driven phase transitions, are discussed in detail. [ABSTRACT FROM AUTHOR]

Hen egg-white lysozyme (HEWL) is a widely used model protein in crystallographic studies and its enzymatic mechanism has been extensively investigated for decades. Despite this, the interaction between the reaction intermediate and the catalytic Asp52, as well as the orientation of Asn44 and Asn46 side chains, remain ambiguous. Here, we report the crystal structures of perdeuterated HEWL and D 2 O buffer-exchanged HEWL from 0.91 and 1.1 Å resolution neutron diffraction data, respectively. These structures were obtained at room temperature and acidic pH, representing the active state of the enzyme. The unambiguous assignment of hydrogen positions based on the neutron scattering length density maps elucidates the roles of Asn44, Asn46, Asn59, and nearby water molecules in the stabilization of Asp52. Additionally, the identification of hydrogen positions reveals unique details of lysozyme's folding, hydrogen (H)/deuterium (D) exchange, and side chain disorder., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The advent of serial crystallography has rejuvenated and popularized room-temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g. large complexes, membrane proteins and viruses) form fragile crystals that are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallization. Many medically relevant samples are expressed in mammalian cell lines, producing a meager quantity of protein that is incompatible with batch crystallization. This can limit the scope of serial crystallography approaches. Direct in situ data collection from a 96-well crystallization plate enables not only the identification of the best diffracting crystallization condition but also the possibility for structure determination under ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallization plates mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility. We applied our iSX approach on a challenging project, autotaxin, a therapeutic target expressed in a stable human cell line, to determine the structure in the lowest-symmetry P1 space group at 3.0 Å resolution. Our in situ data collection strategy provided a complete dataset for structure determination while screening various crystallization conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallization plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons., (open access.)

The polymorphism of human insulin upon pH variation was characterized via X-ray powder diffraction, employing a crystallization protocol previously established for co-crystallization with phenolic derivatives. Two distinct rhombohedral (R3) polymorphs and one cubic (I2 1 3) polymorph were identified with increasing pH, corresponding to the T 6 , T 3 R 3 f and T 2 conformations of insulin, respectively. The structure of the cubic T 2 polymorph was determined via multi-profile stereochemically restrained Rietveld refinement at 2.7 Å resolution. This constitutes the first cubic insulin structure to be determined from crystals grown in the presence of zinc ions, although no zinc binding was observed. The differences of the polycrystalline variant from other cubic insulin structures, as well as the nature of the pH-driven phase transitions, are discussed in detail., (open access.)