Abstract
Direct observation of functional motions in protein structures is highly desirable for understanding how these nanomachineries of life operate at the molecular level. 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. We recently reported a crystal-on-crystal device to facilitate in situ diffraction of protein crystals at room temperature devoid of any sample manipulation. Here an automated serial crystallography platform based on this crystal-on-crystal technology is presented. 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. This platform has been tested extensively using fragile protein crystals. 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 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.
Highlights
Elucidating protein structural dynamics at the molecular level is the key to our fundamental understanding of biochemical reactions and biological processes
Using two rather fragile protein crystals, we demonstrate that this platform enables automated data collection, giving rise to complete crystallographic datasets by combining hundreds or thousands of diffraction images collected from only a few devices
How to effectively deliver a large number of crystal samples to the X-ray beam is at the center of technological development for serial crystallography at both synchrotrons and X-ray freeelectron lasers (XFELs) (Grunbein & Kovacs, 2019; Martiel et al, 2019)
Summary
Elucidating protein structural dynamics at the molecular level is the key to our fundamental understanding of biochemical reactions and biological processes. As the method of choice, crystallography offers superb spatial and temporal resolution for visualizing protein structures at work. 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. Room-temperature diffraction methods, 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). Large conformational changes have been observed at room temperature (Weinert et al, 2019) as well as elevated cryo temperatures above the glass
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