TY - JOUR T1 - Mapping the Conformational Landscape of a Dynamic Enzyme by Multitemperature and XFEL Crystallography JF - bioRxiv DO - 10.1101/016733 SP - 016733 AU - Daniel A. Keedy AU - Lillian R. Kenner AU - Matthew Warkentin AU - Rahel A. Woldeyes AU - Jesse B. Hopkins AU - Michael C. Thompson AU - Aaron S. Brewster AU - Andrew H. Van Benschoten AU - Elizabeth L. Baxter AU - Monarin Uervirojnangkoorn AU - Scott E. McPhillps AU - Jinhu Song AU - Roberto Alonso-Mori AU - James M. Holton AU - William I. Weis AU - Axel T. Brunger AU - S. Michael Soltis AU - Henrik Lemke AU - Ana Gonzalez AU - Nicholas K. Sauter AU - Aina E. Cohen AU - Henry van den Bedem AU - Robert E. Thorne AU - James S. Fraser Y1 - 2015/01/01 UR - http://biorxiv.org/content/early/2015/08/28/016733.abstract N2 - 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 for 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. ER -