Ice formation and solvent nanoconfinement in protein crystals

Abstract

Ice formation within protein crystals is a major obstacle to the cryocrystallographic study of protein structure, and has limited studies of how the structural ensemble of a protein evolves with temperature in the biophysically interesting range from ∼260 K to the protein–solvent glass transition near 200 K. Using protein crystals with solvent cavities as large as ∼70 Å, time-resolved X-ray diffraction was used to study the response of protein and internal solvent during rapid cooling. Solvent nanoconfinement suppresses freezing temperatures and ice-nucleation rates so that ice-free, low-mosaicity diffraction data can be reliably collected down to 200 K without the use of cryoprotectants. Hexagonal ice (Ih) forms in external solvent, but internal crystal solvent forms stacking-disordered ice (Isd) with a near-random stacking of cubic and hexagonal planes. Analysis of powder diffraction from internal ice and single-crystal diffraction from the host protein structure shows that the maximum crystallizable solvent fraction decreases with decreasing crystal solvent-cavity size, and that an ∼6 Å thick layer of solvent adjacent to the protein surface cannot crystallize. These results establish protein crystals as excellent model systems for the study of nanoconfined solvent. By combining fast cooling, intense X-ray beams and fast X-ray detectors, complete structural data sets for high-value targets, including membrane proteins and large complexes, may be collected at ∼220–240 K that have much lower mosaicities and comparable B factors, and that may allow more confident identification of ligand binding than in current cryocrystallographic practice.

Hypothesis for a mechanism of beam-induced motion in cryo-electron microscopy

“…Shi and coworkers reported protein concentrations in the deposited liquid of $2 mg ml À1 or $0.2%(w/w). This is far too small to appreciably modify the glass-transition temperature of the solution (Angell, 2002) or the critical cooling rates for vitrification (Warkentin et al, 2013), and the fraction of solvent within the first two hydration layers of the protein molecules (which is unlikely to crystallize; Moreau et al, 2019) at this concentration is also small. However, the cryo-EM images reported by Shi and coworkers (and those often obtained in general cryo-EM practice) suggest that the final protein concentrations after blotting and evaporation and that are captured in the plunge-cooled samples are much larger than in the original solution.…”

Solvent flows, conformation changes and lattice reordering in a cold protein crystal

“…In cubic apoferritin crystals these solvent cavities have a facecentred cubic arrangement. Theses cavities are larger than those found in roughly 98% of PDB-deposited protein crystal structures (Moreau et al, 2019). A second set of large cavities lies between the spherical shells, having a characteristic size of 65 Å (the distance between the second nearest-neighbour shells) and together containing roughly 65% of the total solvent volume.…”

“…Although the apoferritin crystallization solutions and the solvent present in the crystal cavities contain significant salt concentrations, these only reduce the freezing temperature of the solution by 4 C (Clegg, 1995). Ice formation is dominated by the effects of nanoconfinement by the protein lattice (Moreau et al, 2019) and, at the concentrations of 10%(v/v) and larger used here, by glycerol.…”

“…The key obstacles to such studies have been the formation of diffraction-destroying crystalline ice and the need to use large, crystal-perturbing cryoprotectant concentrations to prevent it. We have recently shown that ice formation inside protein crystals is dramatically suppressed by nanoconfinement (Moreau et al, 2019). Following abrupt cooling, the internal solvent in cryoprotectant-free protein crystals with solvent cavities as large as $70 Å can be maintained in a (supercooled) liquid state at temperatures down to 200 K for a time sufficient to collect a complete data set.…”

Attaining atomic resolution from in situ data collection at room temperature using counter-diffusion-based low-cost microchips

“…However, sample handling remains a major issue. Cryoprotection of samples is another difficulty that is directly related to a loss of diffraction power and crystal quality (Pflugrath, 2015;Moreau et al, 2019). RT data collection, on the other hand, is a clear way to ISSN 2059-7983 # 2020 International Union of Crystallography avoid this procedure-dependence of data quality, as has recently been highlighted and reviewed (Broecker et al, 2018) for the diffraction of crystals in situ in their crystallization device.…”