Purification, characterization and crystallization of the human 80S ribosome

被引:38
|
作者
Khatter, Heena [1 ]
Myasnikov, Alexander G. [1 ]
Mastio, Leslie [1 ]
Billas, Isabelle M. L. [1 ]
Birck, Catherine [1 ]
Stella, Stefano [1 ]
Klaholz, Bruno P. [1 ]
机构
[1] Univ Strasbourg, CBI, Dept Integrated Struct Biol, IGBMC,CNRS,UMR 7104,INSERM,U964, F-67404 Illkirch Graffenstaden, France
基金
欧洲研究理事会;
关键词
CRYO-EM STRUCTURE; CRYSTAL-STRUCTURE; EUKARYOTIC RIBOSOME; ANGSTROM RESOLUTION; CRYOELECTRON MICROSCOPY; STRUCTURAL BASIS; RNA; SUBUNIT; COMPLEX; PROTEIN;
D O I
10.1093/nar/gkt1404
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Ribosomes are key macromolecular protein synthesis machineries in the cell. Human ribosomes have so far not been studied to atomic resolution because of their particularly complex structure as compared with other eukaryotic or prokaryotic ribosomes, and they are difficult to prepare to high homogeneity, which is a key requisite for high-resolution structural work. We established a purification protocol for human 80S ribosomes isolated from HeLa cells that allows obtaining large quantities of homogenous samples as characterized by biophysical methods using analytical ultracentrifugation and multiangle laser light scattering. Samples prepared under different conditions were characterized by direct single particle imaging using cryo electron microscopy, which helped optimizing the preparation protocol. From a small data set, a 3D reconstruction at subnanometric resolution was obtained showing all prominent structural features of the human ribosome, and revealing a salt concentration dependence of the presence of the exit site tRNA, which we show is critical for obtaining crystals. With these well-characterized samples first human 80S ribosome crystals were obtained from several crystallization conditions in capillaries and sitting drops, which diffract to 26 angstrom resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future high-resolution work.
引用
收藏
页数:11
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