A versatile approach to high-density microcrystals in lipidic cubic phase for room-temperature serial crystallography.

IF 6.1 3区 材料科学 Q1 Biochemistry, Genetics and Molecular Biology
Journal of Applied Crystallography Pub Date : 2023-08-18 eCollection Date: 2023-10-01 DOI:10.1107/S1600576723006428
James Birch, Tristan O C Kwan, Peter J Judge, Danny Axford, Pierre Aller, Agata Butryn, Rosana I Reis, Juan F Bada Juarez, Javier Vinals, Robin L Owen, Eriko Nango, Rie Tanaka, Kensuke Tono, Yasumasa Joti, Tomoyuki Tanaka, Shigeki Owada, Michihiro Sugahara, So Iwata, Allen M Orville, Anthony Watts, Isabel Moraes
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Abstract

Serial crystallography has emerged as an important tool for structural studies of integral membrane proteins. The ability to collect data from micrometre-sized weakly diffracting crystals at room temperature with minimal radiation damage has opened many new opportunities in time-resolved studies and drug discovery. However, the production of integral membrane protein microcrystals in lipidic cubic phase at the desired crystal density and quantity is challenging. This paper introduces VIALS (versatile approach to high-density microcrystals in lipidic cubic phase for serial crystallography), a simple, fast and efficient method for preparing hundreds of microlitres of high-density microcrystals suitable for serial X-ray diffraction experiments at both synchrotron and free-electron laser sources. The method is also of great benefit for rational structure-based drug design as it facilitates in situ crystal soaking and rapid determination of many co-crystal structures. Using the VIALS approach, room-temperature structures are reported of (i) the archaerhodopsin-3 protein in its dark-adapted state and 110 ns photocycle intermediate, determined to 2.2 and 1.7 Å, respectively, and (ii) the human A2A adenosine receptor in complex with two different ligands determined to a resolution of 3.5 Å.

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一种用于室温系列晶体学的脂立方相中高密度微晶的通用方法。
系列结晶学已成为研究整体膜蛋白结构的重要工具。在室温下以最小的辐射损伤从微米大小的弱衍射晶体中收集数据的能力为时间分辨研究和药物发现开辟了许多新的机会。然而,以所需的晶体密度和数量在脂质立方相中生产完整的膜蛋白微晶是具有挑战性的。本文介绍了VIALS(用于串行结晶学的脂立方相中高密度微晶的通用方法),这是一种简单、快速、高效的制备数百微升高密度单晶的方法,适用于同步加速器和自由电子激光源的串行X射线衍射实验。该方法还有利于基于结构的合理药物设计,因为它有利于原位晶体浸泡和快速测定许多共晶结构。使用VIALS方法,报道了(i)处于暗适应状态的古菌毒蛋白-3蛋白和110的室温结构 ns光循环中间体,测定为2.2和1.7 Å,和(ii)与两种不同配体的人类A2A腺苷受体复合物,测定分辨率为3.5 Å。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
10.00
自引率
3.30%
发文量
178
审稿时长
4.7 months
期刊介绍: Many research topics in condensed matter research, materials science and the life sciences make use of crystallographic methods to study crystalline and non-crystalline matter with neutrons, X-rays and electrons. Articles published in the Journal of Applied Crystallography focus on these methods and their use in identifying structural and diffusion-controlled phase transformations, structure-property relationships, structural changes of defects, interfaces and surfaces, etc. Developments of instrumentation and crystallographic apparatus, theory and interpretation, numerical analysis and other related subjects are also covered. The journal is the primary place where crystallographic computer program information is published.
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