Room-temperature X-ray crystallography provides unique insights into protein conformational heterogeneity, but obtaining sufficiently large protein crystals is a common hurdle. Serial synchrotron crystallography (SSX) helps to address this hurdle by allowing the use of many medium- to small-sized crystals. Here, a recently introduced serial sample-support chip system has been used to obtain the first SSX structure of a human phosphatase, specifically protein tyrosine phosphatase 1B (PTP1B) in the unliganded (apo) state. In previous apo room-temperature structures, the active site and allosteric sites adopted alternate conformations, including open and closed conformations of the active-site WPD loop and of a distal allosteric site. By contrast, in our SSX structure the active site is best fitted with a single conformation, but the distal allosteric site is best fitted with alternate conformations. This observation argues for additional nuance in interpreting the nature of allosteric coupling in this protein. Overall, our results illustrate the promise of serial methods for room-temperature crystallography, as well as future avant-garde crystallography experiments, for PTP1B and other proteins.
With the advent of AlphaFold, protein structure prediction has attained remarkable accuracy. These achievements resulted from a focus on single static structures. The next frontier in this field involves enhancing our ability to model conformational ensembles, not just the ground states of proteins. Notably, deposited structures result from interpretation of density maps, which are derived from either X-ray crystallography or cryogenic electron microscopy (cryo-EM). These maps represent ensemble averages, reflecting molecules in multiple conformations. Here, we present the latest developments in qFit, an automated computational approach to model protein conformational heterogeneity into density maps. We present algorithmic advancements to qFit, validated by improved Rfree and geometry metrics across a broad and diverse set of proteins. Automated multiconformer modeling holds significant promise for interpreting experimental structural biology data and for generating novel hypotheses linking macromolecular conformational dynamics to function.
Room-temperature X-ray crystallography provides unique insights into protein conformational heterogeneity, but a common hurdle is obtaining sufficiently large protein crystals. Serial synchrotron crystallography (SSX) helps address this hurdle by allowing the use of many medium- to small-sized crystals. We have used a recently introduced serial sample support chip system to obtain the first SSX structure of a human phosphatase, specifically Protein Tyrosine Phosphatase 1B (PTP1B) in the unliganded (apo) state. In previous apo room-temperature structures, the active site and allosteric sites adopted alternate conformations, including open and closed conformations for the active-site WPD loop and for a distal allosteric site. By contrast, in our SSX structure, the active site is best fit with a single conformation, but the distal allosteric site is best fit with alternate conformations. This observation argues for additional nuance in interpreting the nature of allosteric coupling in this protein. Overall, our results illustrate the promise of serial methods for room-temperature crystallography, as well as future avant-garde crystallography experiments, for PTP1B and other proteins.
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