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

Abstract

When protein crystals are abruptly cooled, the unit-cell, protein and solventcavity volumes all contract, but the volume of bulk-like internal solvent may expand. Outflow of this solvent from the unit cell and its accumulation in defective interior crystal regions has been suggested as one cause of the large increase in crystal mosaicity on cooling. It is shown that when apoferritin crystals are abruptly cooled to temperatures between 220 and 260 K, the unit cell contracts, solvent is pushed out and the mosaicity grows. On temperaturedependent timescales of 10 to 200 s, the unit-cell and solvent-cavity volume then expand, solvent flows back in, and the mosaicity and B factor both drop. Expansion and reordering at fixed low temperature are associated with smallamplitude but large-scale changes in the conformation and packing of apoferritin. These results demonstrate that increases in mosaicity on cooling arise due to solvent flows out of or into the unit cell and to incomplete, arrested relaxation of protein conformation. They indicate a critical role for time in variable-temperature crystallographic studies, and the feasibility of probing interactions and cooperative conformational changes that underlie cold denaturation in the presence of liquid solvent at temperatures down to $200 K.

Water Networks Repopulate Protein–Ligand Interfaces with Temperature

“…On the contrary, several high RMSD areas at cryo, for instance near the N‐ and C‐terminus, show no signature at RT. The reduced noise level at RT reveals that the apparent hotspots in cryogenic temperature heatmaps are in fact idiosyncratic cooling artifacts possibly from variable cooling rates [30] rather than due to differences in data quality (Supporting Information Table S4); the resolution of cryo‐fragment structures are comparable (Δ Res ≈0.1 Å). While there are no apparent signatures in the comparison of fragments to drug‐like molecules at cryo, it is of note that the respective RT heatmaps reveal shared signatures even between small and large ligands both inside and outside the binding site (Figure 4A).…”

“…Our data suggest that non‐systematic differences often do not reflect genuine responses to ligand binding but rather cryo‐cooling idiosyncrasies. These often originate from differences in cooling rates as crystals of different sizes traverse a changing layer of liquid nitrogen [30] . Consequently, widespread cryogenic artifacts hide and distort unique signatures of ligand binding and misinform ligand discovery and design.…”

“…These often originate from differences in cooling rates as crystals of different sizes traverse a changing layer of liquid nitrogen. [30] Consequently, widespread cryogenic artifacts hide and distort unique signatures of ligand binding and misinform ligand discovery and design. Our conformational barcode plots and heatmap signatures address this issue by revealing conformational signals in RT datasets that lie hidden in the noise of heterogeneous cryogenic datasets.…”

Determining biomolecular structures near room temperature using X-ray crystallography: concepts, methods and future optimization

“…Well-ordered crystals at room temperature have much smaller mosaicities ($0.01 or less; Shaikevitch & Kam, 1981;Dobrianov et al, 1999) than cryocooled crystals ($0.2 or more) and much narrower distributions of unit-cell sizes within each crystal (Kriminski et al, 2002). Increased crystal disorder at cryogenic temperature results from differences in the thermal contraction of the internal crystal solvent volume, protein volume and unit-cell volume, which drive inhomogeneous redistribution of solvent within the crystal and associated lattice disruptions (Juers & Matthews, 2001, 2004aKriminski et al, 2002;Moreau et al, 2019b); from incomplete relaxation of protein and lattice structure towards their temperature-dependent equilibrium during cooling, leading to quenched heterogeneity (Moreau et al, 2019b); and from crystal bending and cracking caused by external and internal stresses during cryocooling. Cold crystal unit cells and order also depend on the cooling rates (Moreau et al, 2019b), which are poorly controlled and highly variable in current cryocrystallography practice.…”