一种用户友好的即插即用的基于环烯烃共聚物的微流控芯片,用于室温、固定靶序列晶体学。

IF 2.6 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Acta Crystallographica. Section D, Structural Biology Pub Date : 2023-10-01 Epub Date: 2023-09-25 DOI:10.1107/S2059798323007027
Zhongrui Liu, Kevin K Gu, Megan L Shelby, Deepshika Gilbile, Artem Y Lyubimov, Silvia Russi, Aina E Cohen, Sankar Raju Narayanasamy, Sabine Botha, Christopher Kupitz, Raymond G Sierra, Fredric Poitevin, Antonio Gilardi, Stella Lisova, Matthew A Coleman, Matthias Frank, Tonya L Kuhl
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引用次数: 0

摘要

在过去的二十年里,连续的X射线晶体学已经能够确定各种蛋白质的结构。随着X射线自由电子激光器(XFEL)的出现,越来越小的晶体产生了高分辨率的衍射和结构测定。继续推进的一个关键需求是将易碎的微米大小的晶体有效地输送到X射线束的交叉点。本文提出了一种用于室温固定目标串行晶体学的全聚合物微流体“芯片”的改进设计,该芯片可以定制以广泛满足同步加速器或XFEL光源用户的需求。该芯片旨在围绕不同类型的晶体进行定制,为用户提供一个友好、快速、方便、超低成本和强大的样品递送平台。与之前的芯片迭代相比[Gilbile等人(2021),Lab chip,214831-4845],新设计消除了洁净室制造。它具有更大的成像面积比体积,同时保持原位结晶或直接晶浆加载的晶体水合稳定性。使用溶菌酶和thaumatin两种模型蛋白的晶体在同步加速器(溶菌酶和thammatin)和XFEL(仅溶菌酶)设施中验证设计的有效性,产生了分辨率为1.42、1.48和1.70的完整数据集 Å。总的来说,改进的芯片设计、易于制造和高可修改性创造了一种强大的、全方位的样品递送工具,结构生物学家可以快速采用,尤其是在样品体积有限和晶体小而脆弱的情况下。
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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A user-friendly plug-and-play cyclic olefin copolymer-based microfluidic chip for room-temperature, fixed-target serial crystallography.

Over the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip' for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbile et al. (2021), Lab Chip, 21, 4831-4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for both in situ crystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70 Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals.

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来源期刊
Acta Crystallographica. Section D, Structural Biology
Acta Crystallographica. Section D, Structural Biology BIOCHEMICAL RESEARCH METHODSBIOCHEMISTRY &-BIOCHEMISTRY & MOLECULAR BIOLOGY
CiteScore
4.50
自引率
13.60%
发文量
216
期刊介绍: Acta Crystallographica Section D welcomes the submission of articles covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules or the methods used to determine them. Reports on new structures of biological importance may address the smallest macromolecules to the largest complex molecular machines. These structures may have been determined using any structural biology technique including crystallography, NMR, cryoEM and/or other techniques. The key criterion is that such articles must present significant new insights into biological, chemical or medical sciences. The inclusion of complementary data that support the conclusions drawn from the structural studies (such as binding studies, mass spectrometry, enzyme assays, or analysis of mutants or other modified forms of biological macromolecule) is encouraged. Methods articles may include new approaches to any aspect of biological structure determination or structure analysis but will only be accepted where they focus on new methods that are demonstrated to be of general applicability and importance to structural biology. Articles describing particularly difficult problems in structural biology are also welcomed, if the analysis would provide useful insights to others facing similar problems.
期刊最新文献
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