Zhongrui Liu, Kevin Gu, Megan Shelby, Debdyuti Roy, Srinivasan Muniyappan, Marius Schmidt, Sankar Raju Narayanasamy, Matthew Coleman, Matthias Frank, Tonya L Kuhl
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In this article, different designs of user-friendly counter-diffusion chambers are presented which can be used to grow large protein crystals in a 2D polymer microfluidic fixed-target chip. Methods for rapid chip fabrication using commercially available thin-film materials such as Mylar, propyl-ene and Kapton are also detailed. Rules of thumb are provided to tune the nucleation and crystal growth to meet users' needs while minimizing sample consumption. These designs provide a reliable approach to forming large crystals and maintaining their hydration for weeks and even months. This allows ample time to grow, select and preserve the best crystal batches before X-ray beam time. Importantly, the fixed-target microfluidic chip has a low background scatter and can be directly used at beamlines without any crystal handling, enabling crystal quality to be preserved. The approach is demonstrated with serial diffraction of photoactive yellow protein, yielding 1.32 Å resolution at room temperature. Fabrication of this standard microfluidic chip with commercially available thin films greatly simplifies fabrication and provides enhanced stability under vacuum. 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引用次数: 0
摘要
与间歇扩散和蒸汽扩散方法相比,反扩散可以生成更大、更高质量的蛋白质晶体,从而获得更好的衍射数据和更高分辨率的结构。通常情况下,反扩散实验在玻璃毛细管等细长腔体内进行,晶体在毛细管中直接测量,或在 X 射线光束线提取并安装。尽管反扩散蛋白质结晶有很多优点,但利用反扩散结晶的固定目标设备却很少。本文介绍了不同的用户友好型反扩散室设计,可用于在二维聚合物微流控固定靶芯片中生长大型蛋白质晶体。此外,还详细介绍了使用市售薄膜材料(如 Mylar、丙烯和 Kapton)快速制造芯片的方法。提供了经验法则来调整成核和晶体生长,以满足用户的需求,同时最大限度地减少样品消耗。这些设计为形成大晶体并在数周甚至数月内保持其水合状态提供了可靠的方法。这就为在 X 射线束时间到来之前生长、选择和保存最佳晶体批次留出了充足的时间。重要的是,固定目标微流体芯片的背景散射很低,可直接用于光束线,无需处理任何晶体,从而保证了晶体质量。该方法通过光活性黄色蛋白质的序列衍射进行了演示,在室温下可获得 1.32 Å 的分辨率。利用市场上可买到的薄膜制造这种标准微流控芯片,大大简化了制造过程,并提高了真空下的稳定性。这些进步将进一步扩大晶体学家对微流体固定靶的利用。
In situ counter-diffusion crystallization and long-term crystal preservation in microfluidic fixed targets for serial crystallography.
Compared with batch and vapor diffusion methods, counter diffusion can generate larger and higher-quality protein crystals yielding improved diffraction data and higher-resolution structures. Typically, counter-diffusion experiments are conducted in elongated chambers, such as glass capillaries, and the crystals are either directly measured in the capillary or extracted and mounted at the X-ray beamline. Despite the advantages of counter-diffusion protein crystallization, there are few fixed-target devices that utilize counter diffusion for crystallization. In this article, different designs of user-friendly counter-diffusion chambers are presented which can be used to grow large protein crystals in a 2D polymer microfluidic fixed-target chip. Methods for rapid chip fabrication using commercially available thin-film materials such as Mylar, propyl-ene and Kapton are also detailed. Rules of thumb are provided to tune the nucleation and crystal growth to meet users' needs while minimizing sample consumption. These designs provide a reliable approach to forming large crystals and maintaining their hydration for weeks and even months. This allows ample time to grow, select and preserve the best crystal batches before X-ray beam time. Importantly, the fixed-target microfluidic chip has a low background scatter and can be directly used at beamlines without any crystal handling, enabling crystal quality to be preserved. The approach is demonstrated with serial diffraction of photoactive yellow protein, yielding 1.32 Å resolution at room temperature. Fabrication of this standard microfluidic chip with commercially available thin films greatly simplifies fabrication and provides enhanced stability under vacuum. These advances will further broaden microfluidic fixed-target utilization by crystallographers.
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.