异质生化组分的低温电镜分析阐明了来自多细胞嗜热真核生物的多种蛋白质复合物

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Dmitry A. Semchonok , Fotis L. Kyrilis , Farzad Hamdi , Panagiotis L. Kastritis
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引用次数: 0

摘要

生物分子复合物及其相互作用支配着细胞的结构和功能。了解它们的结构是解剖细胞内部工作的先决条件,但它们的高阶组装通常是短暂的,并且对结构分析具有挑战性。在这里,我们对来自嗜热毛毛菌细胞提取物的单一、高度异质的生化部分进行低温电镜观察,以观察多细胞真核生物的生物分子含量。低温电镜单粒子图像处理结果表明,多种化学性质不同的生物大分子同时进行三维结构表征是可行的。即,对(a) ATP柠檬酸解酶,(b) Hsp90, (c) 20S蛋白酶体,(d) Hsp60和(e) udp -葡萄糖焦磷酸化酶的嗜热真核复合体进行了表征。这5种复合物在190.64 μm2的嗜热真核生物中被解剖,其中2种复合物,特别是20S蛋白酶体和Hsp60,表现出侧链分辨率特征。热嗜热杆菌Hsp60近原子模型在3.46 Å (FSC = 0.143)处被分辨出来,显示出其赤道结构域的铰链状构象变化,与之前显示的细菌同源物GroEL高度相似。这项工作表明,细胞提取物的冷冻电镜将大大加快细胞复合物的结构分析,并提供前所未有的机会,以整体的方法来注释生物分子的结构。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Cryo-EM of a heterogeneous biochemical fraction elucidates multiple protein complexes from a multicellular thermophilic eukaryote

Cryo-EM of a heterogeneous biochemical fraction elucidates multiple protein complexes from a multicellular thermophilic eukaryote

Biomolecular complexes and their interactions govern cellular structure and function. Understanding their architecture is a prerequisite for dissecting the cell's inner workings, but their higher-order assembly is often transient and challenging for structural analysis. Here, we performed cryo-EM on a single, highly heterogeneous biochemical fraction derived from Chaetomium thermophilum cell extracts to visualize the biomolecular content of the multicellular eukaryote. After cryo-EM single-particle image processing, results showed that a simultaneous three-dimensional structural characterization of multiple chemically diverse biomacromolecules is feasible. Namely, the thermophilic, eukaryotic complexes of (a) ATP citrate-lyase, (b) Hsp90, (c) 20S proteasome, (d) Hsp60 and (e) UDP-glucose pyrophosphorylase were characterized. In total, all five complexes have been structurally dissected in a thermophilic eukaryote in a total imaged sample area of 190.64 μm2, and two, in particular, 20S proteasome and Hsp60, exhibit side-chain resolution features. The C. thermophilum Hsp60 near-atomic model was resolved at 3.46 Å (FSC = 0.143) and shows a hinge-like conformational change of its equatorial domain, highly similar to the one previously shown for its bacterial orthologue, GroEL. This work demonstrates that cryo-EM of cell extracts will greatly accelerate the structural analysis of cellular complexes and provide unprecedented opportunities to annotate architectures of biomolecules in a holistic approach.

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来源期刊
Journal of Structural Biology: X
Journal of Structural Biology: X Biochemistry, Genetics and Molecular Biology-Structural Biology
CiteScore
6.50
自引率
0.00%
发文量
20
审稿时长
62 days
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