%0 Journal Article %A Daniel A. Keedy %A Lillian R. Kenner %A Matthew Warkentin %A Rahel A. Woldeyes %A Michael C. Thompson %A Aaron S. Brewster %A Andrew H. Van Benschoten %A Elizabeth L. Baxter %A Jesse B. Hopkins %A Monarin Uervirojnangkoorn %A Scott E. McPhillps %A Jinhu Song %A Roberto Alonso-Mori %A James M. Holton %A William I. Weis %A Axel T. Brunger %A S. Michael Soltis %A Henrik Lemke %A Ana Gonzalez %A Nicholas K. Sauter %A Aina E. Cohen %A Henry van den Bedem %A Robert E. Thorne %A James Fraser %T Mapping the Conformational Landscape of a Dynamic Enzyme by XFEL and Multitemperature Crystallography %D 2015 %R 10.1101/016733 %J bioRxiv %P 016733 %X 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. Here we compare the conformational ensembles of CypA by fixed-target X-ray free electron laser (XFEL) crystallography and multitemperature synchrotron crystallography. The “diffraction-before-destruction” nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA. 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 above, but not below, the glass transition temperature (∼200 K) and reveal abrupt changes in protein flexibility that provide all-atom insight into conformational coupling. Together, our XFEL data and multitemperature analyses motivate a new generation of time-resolved experiments to structurally characterize the dynamic underpinnings of protein function. %U https://www.biorxiv.org/content/biorxiv/early/2015/03/19/016733.full.pdf