Data_Sheet_1_Definition of the Minimal Contents for the Molecular Simulation of the Yeast Cytoplasm.pdf

The cytoplasm is a densely packed environment filled with macromolecules with hindered diffusion. Molecular simulation of the diffusion of biomolecules under such macromolecular crowding conditions requires the definition of a simulation cell with a cytoplasmic-like composition. This has been previously done for prokaryote cells (E. coli) but not for eukaryote cells such as yeast as a model organism. Yeast proteomics datasets vary widely in terms of cell growth conditions, the technique used to determine protein composition, the reported relative abundance of proteins, and the units in which abundances are reported. We determined that the gene ontology profiles of the most abundant proteins across these datasets are similar, but their abundances vary greatly. To overcome this problem, we chose five mass spectrometry proteomics datasets that fulfilled the following criteria: high internal consistency, consistency with published experimental data, and freedom from GFP-tagging artifacts. Using these datasets, the contents of a simulation cell containing a single 80S ribosome were defined, such that the macromolecular density and the mass ratio of ribosomal-to-cytoplasmic proteins were consistent with experiment and chosen datasets. Finally, multiple tRNAs were added, consistent with their experimentally-determined number in the yeast cell. The resulting composition can be readily used in molecular simulations representative of yeast cytoplasmic macromolecular crowding conditions to characterize a variety of phenomena, such as protein diffusion, protein-protein interactions and biological processes such as protein translation.

细胞质是一类高度致密拥挤的环境，内部充满大分子且分子扩散存在受阻效应。在这类大分子拥挤条件下开展生物分子扩散的分子模拟研究，需要构建具有类细胞质组成的模拟胞腔。此前已有针对原核细胞（E. coli）的相关研究，但尚未针对以酵母为模式生物的真核细胞完成此类构建。现有酵母蛋白质组学数据集存在显著差异，差异维度涵盖细胞培养条件、蛋白质组成测定技术、已报道的蛋白质相对丰度，以及丰度的计量单位。本研究发现，尽管不同数据集内丰度最高的蛋白质的基因本体（Gene Ontology，GO）注释谱较为相似，但其丰度水平差异显著。为解决这一问题，本研究筛选出符合以下三项标准的5份质谱蛋白质组学数据集：内部一致性高、与已发表实验数据吻合、不存在GFP标签导致的实验伪影。基于上述数据集，本研究明确了包含单个80S核糖体（80S ribosome）的模拟胞腔的组成，使得其中的大分子密度以及核糖体蛋白与胞质蛋白的质量比，均与实验结果及所选数据集的结果保持一致。最后，本研究根据酵母细胞内经实验测定的转运RNA（transfer RNA，tRNA）数量，添加了对应数目的各类tRNA。本研究得到的模拟胞腔组成，可直接用于模拟酵母细胞质大分子拥挤环境的分子模拟研究，以表征多种分子现象，例如蛋白质扩散、蛋白质-蛋白质相互作用，以及蛋白质翻译等生物学过程。