TY - JOUR
T1 - Purification, characterization and crystallization of the human 80S ribosome
AU - Khatter, Heena
AU - Myasnikov, Alexander G.
AU - Mastio, Leslie
AU - Billas, Isabelle M.L.
AU - Birck, Catherine
AU - Stella, Stefano
AU - Klaholz, Bruno P.
PY - 2014/1/1
Y1 - 2014/1/1
N2 - 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 wellcharacterized samples first human 80S ribosome crystals were obtained from several crystallization conditions in capillaries and sitting drops, which diffract to 26Å resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future highresolution work.
AB - 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 wellcharacterized samples first human 80S ribosome crystals were obtained from several crystallization conditions in capillaries and sitting drops, which diffract to 26Å resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future highresolution work.
UR - http://www.scopus.com/inward/record.url?scp=84898979617&partnerID=8YFLogxK
U2 - 10.1093/nar/gkt1404
DO - 10.1093/nar/gkt1404
M3 - Journal article
C2 - 24452798
AN - SCOPUS:84898979617
SN - 0305-1048
VL - 42
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 6
ER -