Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.DOI: http://dx.doi.org/10.7554/eLife.07574.001

The temperature-dependence of radiation damage to thaumatin crystals between T = 300 and 100 K is reported. The amount of damage for a given dose decreases sharply as the temperature decreases from 300 to 220 K and then decreases more gradually on further cooling below the protein-solvent glass transition. Two regimes of temperature-activated behavior were observed. At temperatures above $200 K the activation energy of 18.0 kJ mol À1 indicates that radiation damage is dominated by diffusive motions in the protein and solvent. At temperatures below $200 K the activation energy is only 1.00 kJ mol À1 , which is of the order of the thermal energy. Similar activation energies describe the temperaturedependence of radiation damage to a variety of solvent-free small-molecule organic crystals over the temperature range T = 300-80 K. It is suggested that radiation damage in this regime is vibrationally assisted and that the freezing-out of amino-acid scale vibrations contributes to the very weak temperature-dependence of radiation damage below $80 K. Analysis using the radiation-damage model of Blake and Phillips [Blake & Phillips (1962), Biological Effects of Ionizing Radiation at the Molecular Level, indicates that large-scale conformational and molecular motions are frozen out below T = 200 K but become increasingly prevalent and make an increasing contribution to damage at higher temperatures. Possible alternative mechanisms for radiation damage involving the formation of hydrogen-gas bubbles are discussed and discounted. These results have implications for mechanistic studies of proteins and for studies of the protein glass transition. They also suggest that data collection at T ' 220 K may provide a viable alternative for structure determination when cooling-induced disorder at T = 100 is excessive.

When samples having volumes characteristic of protein crystals are plunge cooled in liquid nitrogen or propane, most cooling occurs in the cold gas layer above the liquid. By removing this cold gas layer, cooling rates for small samples and modest plunge velocities are increased to 1.5 × 10(4) K s(-1), with increases of a factor of 100 over current best practice possible with 10 μm samples. Glycerol concentrations required to eliminate water crystallization in protein-free aqueous mixtures drop from ∼28% w/v to as low as 6% w/v. These results will allow many crystals to go from crystallization tray to liquid cryogen to X-ray beam without cryoprotectants. By reducing or eliminating the need for cryoprotectants in growth solutions, they may also simplify the search for crystallization conditions and for optimal screens. The results presented here resolve many puzzles, such as why plunge cooling in liquid nitrogen or propane has, until now, not yielded significantly better diffraction quality than gas-stream cooling.

Small angle x-ray scattering (SAXS) is a versatile and widely used technique for obtaining low-resolution structures of macromolecules and complexes. SAXS experiments measure molecules in solution, without the need for labeling or crystallization. However, radiation damage currently limits the application of SAXS to molecules that can be produced in microgram quantities; for typical proteins, 10-20 μL of solution at 1 mg/mL is required to accumulate adequate signal before irreversible x-ray damage is observed. Here, we show that cryocooled proteins and nucleic acids can withstand doses at least two orders of magnitude larger than room temperature samples. We demonstrate accurate T = 100 K particle envelope reconstructions from sample volumes as small as 15 nL, a factor of 1000 smaller than in current practice. Cryo-SAXS will thus enable structure determination of difficult-to-express proteins and biologically important, highly radiation-sensitive proteins including light-activated switches and metalloenzymes.

A series of studies that provide a consistent and illuminating picture of global radiation damage to protein crystals, especially at temperatures above ∼200 K, are described. The radiation sensitivity shows a transition near 200 K, above which it appears to be limited by solvent-coupled diffusive processes. Consistent with this interpretation, a component of global damage proceeds on timescales of several minutes at 180 K, decreasing to seconds near room temperature. As a result, data collection times of order 1 s allow up to half of global damage to be outrun at 260 K. Much larger damage reductions near room temperature should be feasible using larger dose rates delivered using microfocused beams, enabling a significant expansion of structural studies of proteins under more nearly native conditions.