Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein.
Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein.
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DOI：
10.1038/srep30909
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发表时间：
2016-08-03
影响因子：
4.6
通讯作者：
Petosa C
中科院分区：
文献类型：
--
作者：
Coscia F;Estrozi LF;Hans F;Malet H;Noirclerc-Savoye M;Schoehn G;Petosa C
最近的技术进步已经彻底改变了冷冻电子显微镜（cryo-EM）领域。然而，大多数单体蛋白仍然太小（<100 kDa），无法进行低温电镜分析。为了克服这一限制，我们探索了一种策略，即将单体靶蛋白与同源寡聚支架蛋白基因融合，并优化连接以使目标蛋白采用支架对称，从而产生适合冷冻电镜的嵌合粒子。为了证明这一概念，我们将40 kDa单体麦糖结合蛋白（MBP）与谷氨酰胺合成酶（由两个六聚环形成的十二聚体）融合。通过生物物理分析和负染色电镜筛选了不同连接长度的嵌合结构，获得了亚纳米分辨率的MBP结构。这些发现说明了使用同质寡聚支架对单体蛋白进行低温电镜分析的可行性，为将该策略应用于抵抗晶体学和核磁共振分析的结构挑战铺平了道路。
Recent technical advances have revolutionized the field of cryo-electron microscopy (cryo-EM). However, most monomeric proteins remain too small (<100 kDa) for cryo-EM analysis. To overcome this limitation, we explored a strategy whereby a monomeric target protein is genetically fused to a homo-oligomeric scaffold protein and the junction optimized to allow the target to adopt the scaffold symmetry, thereby generating a chimeric particle suitable for cryo-EM. To demonstrate the concept, we fused maltose-binding protein (MBP), a 40 kDa monomer, to glutamine synthetase, a dodecamer formed by two hexameric rings. Chimeric constructs with different junction lengths were screened by biophysical analysis and negative-stain EM. The optimal construct yielded a cryo-EM reconstruction that revealed the MBP structure at sub-nanometre resolution. These findings illustrate the feasibility of using homo-oligomeric scaffolds to enable cryo-EM analysis of monomeric proteins, paving the way for applying this strategy to challenging structures resistant to crystallographic and NMR analysis.