Search

A new approach to in situ experimental phasing introduced by Lawrence et al. (2020, Acta Cryst. D76, 790–801) will be helpful for the macromolecular crystallography community.

By far most macromolecular crystallographic data collection and experimental phasing is nowadays carried out using synchrotron radiation. Here, we present two crystallographic datasets collected on a home-source X-ray diffractometer, which can per se be use to experimentally solve the atomic-resolution crystal structure of the Src homology 3(SH3)-like domain from the postsynaptic protein Shank3. The refined structure was described in the article “Structure of an unconventional SH3 domain from the postsynaptic density protein Shank3 at ultrahigh resolution” (Ponna et al., 2017) [1]. Crystals of the Shank3 SH3 domain were derivatized through soaking in 1 M sodium iodide prior to diffraction data collection at a wavelength of 1.54 Å. High-resolution data are reported for a native crystal to 1.01 Å and an iodide-derivatized one to 1.60 Å. The crystals suffered from several anomalies affecting experimental phasing: a high fraction (34–40%) of pseudomerohedral twinning, significant pseudotranslational symmetry (> 15%) with the operator 0.5,0,0.5, and a low solvent content. Twinning with the operator h,-k,-l is made possible by the space group P21 coupled with a unit cell β angle of 90.0°. The data can be used to repeat and optimize derivatization and phasing procedures, to understand halide interactions with protein surfaces, to promote the use of home X-ray sources for protein structure determination, as well as for educational purposes and protocol development.

For the purpose of this article, experimental phasing is understood to mean the determination of macromolecular structures by exploiting small intensity differences of Friedel opposites and possibly of reflections measured at different wavelengths or for heavy-atom derivatives, without the use of specific structural models. The SHELX programs provide a robust and efficient route for routine structure solution by the SAD, MAD and related methods, but involve a number of simplifying assumptions that may limit their applicability in borderline cases. The substructure atoms (i.e. those with significant anomalous scattering) are first located by direct methods, and the experimental data are then used to estimate phase shifts that are added to the substructure phases to obtain starting phases for the native reflections. These are then improved by density modification and, if the resolution of the data and the type of structure permit, polyalanine tracing. A number of extensions to the tracing algorithm are discussed; these are designed to improve its performance at low resolution. Given native data to 2.5 Å resolution or better, a correlation coefficient greater than 25% between the structure factors calculated from such a trace and the native data is usually a good indication that the structure has been solved.

We present datasets that can be used for the experimental phasing of crystal structures of two myelin proteins. The structures were recently described in the articles “Periaxin and AHNAK nucleoprotein 2 form intertwined homodimers through domain swapping” (H. Han, P. Kursula, 2014) [1] and “The olfactomedin domain from gliomedin is a β-propeller with unique structural properties” (H. Han, P. Kursula, 2015) [2]. Crystals of periaxin were derivatized with tungsten and xenon prior to data collection, and diffraction data for these crystals are reported at 3 and 1 wavelengths, respectively. Crystallographic data for two different pressurizing times for xenon are provided. Gliomedin was derivatized with platinum, and data for single-wavelength anomalous dispersion are included. The data can be used to repeat the phasing experiments, to analyze heavy atom binding sites in proteins, as well as to optimize future derivatization experiments of protein crystals with these and other heavy-atom compounds.

Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins., SCOPUS: ar.j, info:eu-repo/semantics/published