A drug discovery-oriented non-invasive protocol for protein crystal cryoprotection by dehydration, with application for crystallization screening.

In X-ray macromolecular crystallography, cryoprotection of crystals mounted on harvesting loops is achieved when the water in the sample solvent transitions to vitreous ice before crystalline ice forms. This is achieved by rapid cooling in liquid nitrogen or propane. Protocols for protein crystal cryoprotection are based on either increasing environmental pressure or reducing the water fraction in the solvent. This study presents a new protocol for cryoprotecting crystals. It is based on vapour diffusion dehydration of the crystal drop to reduce the water fraction in the solvent by adding a highly concentrated salt solution, 13 M potassium formate (KF13), directly to the reservoir. Cryoprotection by the KF13 protocol is non-invasive to the crystal, high throughput, not labour intensive, can benefit diffraction resolution and ligand binding, and is very useful in cases with high redundancy such as drug discovery projects which utilize very large compound or fragment libraries. Moreover, an application of KF13 to discover new crystal hits from clear drops of equilibrated crystallization screening plates is also shown.

Experimental evidence for the benefits of higher X-ray energies for macromolecular crystallography

X-ray-induced radiation damage is a limiting factor for the macromolecular crystallographer and data must often be merged from many crystals to yield complete data sets for the structure solution of challenging samples. Increasing the X-ray energy beyond the typical 10–15 keV range promises to provide an extension of crystal lifetime via an increase in diffraction efficiency. To date, however, hardware limitations have negated any possible gains. Through the first use of a cadmium telluride EIGER2 detector and a beamline optimized for high-energy data collection, it is shown that at higher energies fewer crystals will be required to obtain complete data, as the diffracted intensity per unit dose increases by a factor of more than two between 12.4 and 25 keV. Additionally, these higher energy data can provide more information, as shown by a systematic increase in the high-resolution cutoff of the data collected. Taken together, these gains point to a high-energy future for synchrotron-based macromolecular crystallography.

BL-11C Micro-MX: a high-flux microfocus macromolecular-crystallography beamline for micrometre-sized protein crystals at Pohang Light Source II

BL-11C, a new protein crystallography beamline, is an in-vacuum undulator-based microfocus beamline used for macromolecular crystallography at the Pohang Accelerator Laboratory and it was made available to users in June 2017. The beamline is energy tunable in the range 5.0–20 keV to support conventional single- and multi-wavelength anomalous-dispersion experiments against a wide range of heavy metals. At the standard working energy of 12.659 keV, the monochromated beam is focused to 4.1 µm (V) × 8.5 µm (H) full width at half-maximum at the sample position and the measured photon flux is 1.3 × 1012 photons s−1. The experimental station is equipped with a Pilatus3 6M detector, a micro-diffractometer (MD2S) incorporating a multi-axis goniometer, and a robotic sample exchanger (CATS) with a dewar capacity of 90 samples. This beamline is suitable for structural determination of weakly diffracting crystalline substances, such as biomaterials, including protein, nucleic acids and their complexes. In addition, serial crystallography experiments for determining crystal structures at room temperature are possible. Herein, the current beamline characteristics, technical information for users and some recent scientific highlights are described.