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X-ray transparent microfluidic platforms for membrane protein crystallization with microseeds†
Lab on a Chip ( IF 6.1 ) Pub Date : 2018-02-08 00:00:00 , DOI: 10.1039/c7lc01141e Jeremy M Schieferstein 1 , Ashtamurthy S Pawate , Michael J Varel , Sudipto Guha , Ieva Astrauskaite , Robert B Gennis , Paul J A Kenis
Lab on a Chip ( IF 6.1 ) Pub Date : 2018-02-08 00:00:00 , DOI: 10.1039/c7lc01141e Jeremy M Schieferstein 1 , Ashtamurthy S Pawate , Michael J Varel , Sudipto Guha , Ieva Astrauskaite , Robert B Gennis , Paul J A Kenis
Affiliation
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Crystallization of membrane proteins is a critical step for uncovering atomic resolution 3-D structures and elucidating structure–function relationships. Microseeding, the process of transferring sub-microscopic crystal nuclei from initial screens into new crystallization experiments, is an effective, yet underutilized approach to grow crystals suitable for X-ray crystallography. Here, we report simplified methods for crystallization of membrane proteins that utilize microseeding in X-ray transparent microfluidic chips. First, a microfluidic method for introduction of microseed dilutions into metastable crystallization experiments is demonstrated for photoactive yellow protein and cytochrome bo3 oxidase. As microseed concentration decreased, the number of crystals decreased while the average size increased. Second, we demonstrate a microfluidic chip for microseed screening, where many crystallization conditions were formulated on-chip prior to mixing with microseeds. Crystallization composition, crystal size, and diffraction data were collected and mapped on phase diagrams, which revealed that crystals of similar diffraction quality and size typically grow in distinct regions of the phase diagram.
中文翻译：
用于使用微种子进行膜蛋白结晶的 X 射线透明微流体平台†
膜蛋白的结晶是揭示原子分辨率的 3-D 结构和阐明结构-功能关系的关键步骤。微晶种是将亚显微晶核从初始筛选转移到新的结晶实验的过程，是一种有效但未充分利用的生长适合 X 射线晶体学的晶体的方法。在这里，我们报告了在 X 射线透明微流控芯片中利用微接种的膜蛋白结晶的简化方法。首先，针对光活性黄色蛋白和细胞色素bo 3氧化酶，演示了将微种子稀释液引入亚稳态结晶实验的微流体方法。随着微晶种浓度的降低，晶体数量减少，而平均尺寸增加。其次，我们展示了一种用于微种子筛选的微流控芯片，其中在与微种子混合之前在芯片上制定了许多结晶条件。收集结晶成分、晶体尺寸和衍射数据并将其绘制在相图上，这表明具有相似衍射质量和尺寸的晶体通常生长在相图的不同区域。
更新日期：2018-02-08
中文翻译：
用于使用微种子进行膜蛋白结晶的 X 射线透明微流体平台†
膜蛋白的结晶是揭示原子分辨率的 3-D 结构和阐明结构-功能关系的关键步骤。微晶种是将亚显微晶核从初始筛选转移到新的结晶实验的过程，是一种有效但未充分利用的生长适合 X 射线晶体学的晶体的方法。在这里，我们报告了在 X 射线透明微流控芯片中利用微接种的膜蛋白结晶的简化方法。首先，针对光活性黄色蛋白和细胞色素bo 3氧化酶，演示了将微种子稀释液引入亚稳态结晶实验的微流体方法。随着微晶种浓度的降低，晶体数量减少，而平均尺寸增加。其次，我们展示了一种用于微种子筛选的微流控芯片，其中在与微种子混合之前在芯片上制定了许多结晶条件。收集结晶成分、晶体尺寸和衍射数据并将其绘制在相图上，这表明具有相似衍射质量和尺寸的晶体通常生长在相图的不同区域。
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