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An automated platform for in situ serial crystallography at room temperature.

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.

SUBMITTER: Ren Z

PROVIDER: S-EPMC7642789 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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An automated platform for <i>in situ</i> serial crystallography at room temperature.

Ren Zhong Z Wang Cong C Shin Heewhan H Bandara Sepalika S Kumarapperuma Indika I Ren Michael Y MY Kang Weijia W Yang Xiaojing X

IUCrJ 20200919 Pt 6

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 <i>in situ</i> diffraction of protein cr ...[more]

PMID: 33209315

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Project description:Summary Membrane proteins are central to many pathophysiological processes, yet remain very difficult to analyze structurally. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperatures. We apply this approach to human integral membrane proteins, which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting, and serial crystallography, enabling screening of small-molecule libraries with membrane protein crystals grown in meso. This approach brings needed automation to this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins. Graphical abstract Highlights • A platform for rapid in meso structures by serial crystallography (SSX)• Insights into ADIPOR2 receptor-ligand dynamic interactions• A web-based application for remote user-guided experimental design and execution• An automated SSX-based ligand discovery pipeline for membrane proteins is introduced Motivation Membrane proteins are key regulators of most physiological processes and represent attractive targets for drug discovery programs. One of the most successful methods to obtain high-resolution structures of membrane proteins relies on in meso crystallization in combination with serial synchrotron crystallography. However, this remains a difficult process, with challenges at every step, including complex manual sample recovery protocols leading to limited throughput and sample loss and the difficulty in carrying out high-throughput ligand screening experiments. We have developed a new approach enabling rapid, automated structural analysis of membrane proteins in meso based on the CrystalDirect technology that addresses these issues, enabling high-throughput drug discovery with membrane proteins. Membrane proteins control many biological processes and represent attractive targets for drug discovery, but are difficult to study structurally. Healey et al. present an automated approach, combining the CrystalDirect technology and serial crystallography, for rapid structural analysis of membrane proteins and opening new opportunities for high-throughput drug discovery.

Dataset Information

An automated platform for in situ serial crystallography at room temperature.

Publications

An automated platform for <i>in situ</i> serial crystallography at room temperature.

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