National Center for In-situ Tomographic Ultramicroscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, United States of America.
Department of Chemistry and Biochemistry, University of Alabama, Tuscaloosa, AL, United States of America.
PLoS One. 2023 Jun 23;18(6):e0287461. doi: 10.1371/journal.pone.0287461. eCollection 2023.
Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex from S. epidermidis bound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 Å density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between the S. epidermidis Cas10-Csm structure and the well-resolved bacterial (S. thermophilus) and archaeal (T. onnurineus) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes.
III 型 CRISPR-Cas 系统利用结合了小 CRISPR RNA（crRNA）的多蛋白效应复合物来检测外来 RNA 转录本，并引发复杂的免疫反应，导致入侵 RNA 和 DNA 的破坏。III 型系统在自然界中分布最广泛，人们对利用这些系统进行生物技术应用的兴趣日益浓厚，这凸显了对来自不同生物体的代表进行详细结构分析的必要性。我们对来自表皮葡萄球菌的 III-A 型 Cas10-Csm 效应复合物与完整的、同源靶 RNA 结合的复合物进行了低温电子显微镜（cryo-EM）重建，并鉴定出两种聚合状态，即 276 kDa 复合物和 318 kDa 复合物。有序的 276 kDa 复合物的 3.1 Å 密度允许在复合物内构建 Csm2、Csm3、Csm4 和 Csm5 亚基以及 crRNA 和靶 RNA 的原子模型。我们还收集了小角度 X 射线散射数据，该数据与我们鉴定的 276 kDa Cas10-Csm 结构一致。表皮葡萄球菌 Cas10-Csm 结构与分辨率较高的细菌（嗜热链球菌）和古菌（火球菌）Cas10-Csm 结构之间的详细比较揭示了复合物与靶 RNA 和 crRNA 相互作用的差异，这些差异可能具有功能意义。这些结构比较揭示了来自不同生物体的 III-A 型系统的独特特征，并将有助于改进源自 III-A 型效应复合物的生物技术。
