Preparation of Doublet Microtubule Fraction for Single Particle Cryo-electron Microscopy
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Bibliographic record
Abstract
Over the years, studying the ultrastructure of the eukaryotic cilia/flagella using electron microscopy (EM) has contributed significantly toward our understanding of ciliary function. Major complexes in the cilia, such as inner and outer dynein arms, radial spokes, and dynein regulatory complexes, were originally discovered by EM. Classical resin-embedding EM or cryo-electron tomography can be performed directly on the isolated cilia or in some cases, cilia directly attached to the cell body. Recently, single particle cryo-EM has emerged as a powerful structural technique to elucidate high-resolution structures of macromolecular complexes; however, single particle cryo-EM requires non-overlapping complexes, i.e., the doublet microtubule of the cilia. Here, we present a protocol to separate the doublet microtubule from the isolated cilia bundle of two species, Tetrahymena thermophila and Chlamydomonas reinhardtii, using ATP reactivation and sonication. Our approach produces good distribution and random orientation of the doublet microtubule fragments, which is suitable for single particle cryo-EM analysis., [摘要]多年来，使用电子显微镜（EM）研究真核纤毛/鞭毛的超微结构为我们对睫毛功能的理解做出了重要贡献。在纤毛主要络合物，如内，外动力蛋白臂，径向轮辐，而动力蛋白调控复合物，是最初由EM发现。可以在分离的纤毛上直接进行经典的树脂包埋EM或冷冻电子断层扫描，或者在某些情况下，可以直接将纤毛直接附着在细胞体上。最近，单粒子冷冻电镜已成为一个结构的强有力的技术，以阐明高分辨率结构小号大分子复合物; ħ H但是，单粒子冷冻电镜需要非重叠的复合物，即，纤毛的双峰微管。在这里，我们提出了一个协议到双峰微管从两个物种的分离的纤毛束分离，四膜虫嗜热和衣藻，使用ATP激活和超声处理。我们的方法可以产生良好的双峰微管片段分布和随机取向，适用于单颗粒冷冻-EM分析。[背景]纤毛是存在的细胞的表面上的毛发状细胞器，并且负责运动和感觉功能。纤毛由一束9个双态微管组成，在活动性纤毛的情况下，纤毛围绕着两个中央单态微管。纤毛包含数百种独特的蛋白质分子。由于这种复杂性，纤毛的结构生物学主要依赖于完整纤毛的分离（Craige等，2013； Gaertig等，2013）。在过去的15年中，借助子图平均技术进行的冷冻电子断层扫描一直是推动完整的真核纤毛分子结构的主要力量（Bui等人，2008; Heuser等人，2009; Imhof等人，2019 ）。然而，低温电子断层扫描的分辨率通常被设定在范围内的20 -40埃，这限制的能力创建的纤毛蛋白的精确原子模型或相互作用小号对于其功能的机制理解不同蛋白质之间。最近，在前进的单粒子低温电子显微镜（低温EM）技术已允许编高-分辨率结构小号大分子复合物典型地在3-4埃范围内的; ħ H但是，单粒子冷冻电镜不与整个工作纤毛由于重叠的二重峰的微管。我们针对冷冻EM的双峰微管纯化和分级分离方法通过将双峰微管从纤毛中分离出来，克服了这一局限性。这促进了通过再活化动力蛋白利用ATP，超声处理，和盐提取（图1） ，这产生良好-适用于单粒子冷冻电镜的双重微管的分离的片段（市川等人，2017）（图2） 。实际上，这允许将衣藻和四膜虫的双胞胎微管结构重建到3-4Å分辨率，并使双胞胎内的微管蛋白可视化（Ichikawa等人，2019年; Khalifa等人，2020年）。此外，这些双峰微管纯化方案适合于蛋白质组学analys Ë中心对和微管内的蛋白质是因为它们所有的外表面和内动力蛋白臂组件的盐洗涤过程中富集的那些蛋白质（戴等人。，20 20）。 图1.cryo-EM样品制备的工作流程图2.四膜虫（A）和衣藻（B）双峰片段的Cryo-EM图像。比例尺：50 nm。
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from it