However, since protein crystals are extremely sensitive to X-ray radiation damage at room temperature, serial protocols must be adopted to collect X-ray diffraction data from a large number of crystals. Surprisingly large conformational changes have been observed at room temperature (Weinert et al., 2019 ) as well as elevated cryo temperatures above the glass transition point (Bandara et al., 2017 Shin et al., 2019 Zeng et al., 2015 ). Renewed interests in recent years have been inspired by the great promises of short X-ray pulses from polychromatic synchrotron beamlines and X-ray free-electron lasers (XFELs) in protein dynamics studies (Kupitz et al., 2014 Nango et al., 2016 Nogly et al., 2018 Stellato et al., 2014 Tenboer et al., 2014 ). On the other hand, room-temperature diffraction methods, although highly desirable for dynamic studies, have experienced arrested development over the past decades since cryocrystallography became the gold standard for protein structure determination (Shoemaker & Ando, 2018 ). Despite its highly streamlined workflow, protein crystallography has been largely limited to cryogenic conditions that inherently deter large-amplitude protein motions and impede the introduction of structural perturbations for dynamic studies. As the method of choice, crystallography offers superb spatial and temporal resolution for visualizing protein structures at work. This serial data collection platform is compatible with both monochromatic oscillation and Laue methods for X-ray diffraction and presents a widely applicable approach for static and dynamic crystallographic studies at room temperature.Įlucidating protein structural dynamics at the molecular level is the key to our fundamental understanding of biochemical reactions and biological processes. We demonstrate that with affordable sample consumption, this in situ serial crystallography technology could give rise to room-temperature protein structures of higher resolution and superior map quality for those protein crystals that encounter difficulties during freezing. This platform has been tested extensively using fragile protein crystals.
Cryogenic temp xray diffraction software#
A hardware and software prototype has been implemented, and protocols have been established that allow users to image, recognize and rank hundreds to thousands of protein crystals grown on a chip in optical scanning mode prior to serial introduction of these crystals to an X-ray beam in a programmable and high-throughput manner. Here an automated serial crystallography platform based on this crystal-on-crystal technology is presented. We recently reported a crystal-on-crystal device to facilitate in situ diffraction of protein crystals at room temperature devoid of any sample manipulation. Because cryogenic temperatures are non-physiological and may prohibit or even alter protein structural dynamics, it is necessary to develop robust X-ray diffraction methods that enable routine data collection at room temperature. Direct observation of functional motions in protein structures is highly desirable for understanding how these nanomachineries of life operate at the molecular level.
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