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On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies

机译：片上结晶用于串行晶体学实验和片上配体结合研究

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Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, `naked' crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.
机译：有效和可靠的样品输送仍然是串行晶体学实验的瓶颈之一。与其他方法相比，固定目标样品的交付具有以下优势：由于命中率较高，因此可以显着减少样品消耗并缩短数据收集时间。在这里，报道了一种新的片上结晶方法，该方法允许在微图案化的硅芯片上直接有效且可重现地生长大量蛋白质晶体，以进行原位串行晶体学实验。通过坐滴式蒸汽扩散来生长晶体，并且可以直接应用先前建立的结晶条件。通过减少晶体处理步骤的数量，该方法特别适用于敏感的晶体系统。通过吸墨可以有效地从晶体中除去过量的母液，并且不需要密封固定目标样品架以防止晶体脱水。结果，在芯片上获得了“裸”晶体，从而导致非常低的背景散射水平，并使该晶体非常易于外部操作（例如配体溶液的施加）使用。在同步加速器的低温和室温的X射线自由电子激光下进行的系列衍射实验产生了人膜蛋白水通道蛋白2的高质量X射线结构，以及嗜热菌素和水溶蛋白的两个新的配体结合结构。人激酶DRAK2。结果突出了该方法在未来高通量药物化合物芯片上筛选的适用性。

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