Towards the Visual Proteomics of C. reinhardtii using High-throughput Collaborative in situ Cryo-ET.
Towards the Visual Proteomics of C. reinhardtii using High-throughput Collaborative in situ Cryo-ET.
复制标题
使用高通量协作原位冷冻电子断层扫描 (Cryo-ET) 实现莱茵衣藻的视觉蛋白质组学。
DOI：
10.1093/micmic/ozad067.480
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Kotecha,Abhay
中科院分区：
文献类型：
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作者：
Khavnekar,Sagar;Kelley,Ron;Waltz,Florent;Wietrzynski,Wojciech;Zhang,Xianjun;Obr,Martin;Tagiltsev,Grigory;Beck,Florian;Wan,William;Briggs,John;Engel,Ben;Plitzko,Juergen;Kotecha,Abhay
低温电子断层扫描（cryo-ET）正在成为确定生物大分子原位结构的首选方法，即在细胞内或细胞外颗粒的天然环境中。近年来，cryo-ET和随后的分子量在兆道尔顿数量级的大型大分子复合物的亚层析成像平均已经产生了足以显示侧链的分辨率的结构（3-5Å）。这些标本通常冷冻固定在200纳米或更薄的玻璃化冰中。较厚的玻璃化单细胞和最近来自多细胞生物的组织已经使用镓低温聚焦离子束（Ga+-低温fib铣削）进行微机械加工，以生产厚度为200纳米或更小的电子透明切片；这些部分被称为薄片。尽管足够薄，但这些低温fib研磨薄片内大分子复合物的亚层析成像平均值通常限制在低于10 Å的分辨率。这些分辨率限制可能是由于倾斜序列数据采集过程中充电引起的再沉积和光束诱导运动导致的片层质量差，因为Ga+- cro - fib硬件和协议的最新改进使大分子结构的亚层析成像平均分辨率低于5 Å。此外，由于手工样品转移和Ga+离子束损伤，目前的Ga+-冷冻fib系统的吞吐量较低。赛默飞世尔科技公司（Thermo Fisher Scientific）的新一代冷冻fib使用等离子体代替镓（cryo-plasmaFIB），减少了再沉积和离子束损伤，同时通过自动样品转移提高了吞吐量。结合倾斜序列数据采集自动化的最新进展，现在可以收集细胞和组织薄片上的大量原位冷冻et数据集，以确定高分辨率的分子图谱，即视觉蛋白质组学。为了对cryo-plasmaFIB的性能进行基准测试，我们以酿酒葡萄球菌作为建立的模型体系。初步的基准测试结果显示，在单个薄片上获得的7张断层图中，80S核糖体的亚断层图平均为6.5 Å，使用氙等离子体进行低温fib研磨（图1A）。除了可以忽略不计的再沉积外，自动转移带来的吞吐量增加允许更快的筛选，从而形成大型数据集。在对包含260个层析图（119k个子卷）的整个数据集进行分析后，我们能够获得大亚单元的sub-5Å子层析图平均值，其局部分辨率扩展到Nyquist分辨率（图1B）。我们对不同厚度薄片中80S核糖体的Rosenthal-Henderson b因子的分析表明，样品厚度与信噪比（SNR）呈反比关系（图1C）。虽然这可能表明它有利于微机械薄片，但进一步分析片层内不同深度的80S核糖体的Rosenthal-Henderson b因子表明大约有30纳米（nm）的损伤层（图1D）。这些基准结果证明了新开发的cryo-plasmaFIB在高通量和高分辨率细胞cryo-ET中实现视觉蛋白质组学方法的潜力。作为这种方法原理的证明，我们目前正在生成一个大型的原位冷冻- et数据集，其中包含来自C. reinhardtii的数千张断层图。C. reinhardtii提供了一个完善的模型系统来研究许多基本的细胞过程（图2A），并影响了我们对人类疾病的理解。然而，先前的研究仅限于低分辨率（bbb10 Å）亚层图。
Cryo-electron tomography (cryo-ET) is emerging as the method of choice for determining the structures of biological macromolecules in situ, that is, within their native context inside cells or extracellular particles. In recent years, cryo-ET and subsequent subtomogram averaging of large macromolecular complexes with molecular weights on the order of megadaltons have produced structures at resolutions sufficient to visualize sidechains (3-5Å). These specimens have typically been cryo-fixed in vitrified ice 200nm or thinner. Thicker vitrified single cells and, more recently, tissues from multicellular organisms have been micromachined using Gallium cryo-focused ion beam (Ga+-cryo-FIB milling) to produce electron transparent sections with thicknesses of 200 nanometres or less; these sections are referred to as lamellae. Despite being sufficiently thin, subtomogram averages of macromolecular complexes within these cryo-FIB milled lamellae have often been limited to resolutions lower than 10 Å. These resolution limitations were likely due to poor lamella quality owning to redeposition and beam induced motion due to charging during tilt-series data acquisition, as recent improvements in Ga+-cryo-FIB hardware and protocols have enabled subtomogram averaging of macromolecular structures at sub-5 Å resolutions. Furthermore, current Ga+-cryo-FIB systems suffer from low throughput due to manual sample transfers and Ga+ ion beam damage. A next-generation cryo-FIB that uses plasma instead of Gallium (cryo-plasmaFIB), from Thermo Fisher Scientific reduces redeposition and ion beam damage while improving the throughput with automated sample transfers. Combined with recent advances in automation of tilt-series data acquisition, it is now possible to collect large in situ cryo-ET datasets on lamellae of cells and tissues to determine high-resolution molecular atlases, ie, visual proteomics. In order to benchmark the performance of the cryo-plasmaFIB, we used S. cerevisiae as an established model system. Preliminary benchmarks resulted in 6.5 Å subtomogram average of 80S ribosomes from 7 tomograms acquired on a single lamella, cryo-FIB milled using Xenon plasma (Figure 1A). In addition to negligible redeposition, increased throughput from automated transfers allowed for faster screening, leading to a large dataset. Upon analysis of the whole dataset containing 260 tomograms (119k subvolumes), we were able to obtain a sub-5Å subtomogram average of the large subunit with local resolution extending to the Nyquist resolution (Figure 1B). Our analysis of Rosenthal-Henderson B-factors of 80S ribosomes from lamellae with varying thicknesses demonstrates an inverse relationship between specimen thickness and signal-to-noise ratio (SNR)(Figure 1C). While this may suggest that it is beneficial to micromachine thinner lamellae, further analysis of Rosenthal-Henderson B-factors for 80S ribosomes at varying depth within lamellae indicates approximately 30 nanometre (nm) damage layer (Figure 1D). These benchmark results demonstrate the potential of newly developed cryo-plasmaFIB for high-throughput and high-resolution cellular cryo-ET to realise visual proteomics approaches. As a proof of principle of such approaches, we are currently generating a large in situ cryo-ET dataset containing thousands of tomograms from C. reinhardtii. C. reinhardtii offers a well-established model system to study many fundamental cellular processes (Figure 2A) and has impacted our understanding of human diseases. However, prior studies to map macromolecular complexes in C. reinhardtii were limited to lower resolution (> 10 Å) subtomogram …