Purification, characterization and crystallization of the human 80S ribosome.
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 Å resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future high-resolution work.
Centre for Integrative Biology (CBI), Department of Integrated Structural Biology, IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale (INSERM) U964/Université de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch, France.
Heena
H
Khatter
Alexander G
AG
Myasnikov
Leslie
L
Mastio
Isabelle M L
IM
Billas
Catherine
C
Birck
Stefano
S
Stella
Bruno P
BP
Klaholz
D002458:Cell Fractionation / Q000379:methods; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D012270:Ribosomes / Q000737:chemistry* / Q000648:ultrastructure