Purification, characterization and crystallization of the human 80S ribosome

Heena Khatter; Alexander G. Myasnikov; Leslie Mastio; Isabelle M.L. Billas; Catherine Birck; Stefano Stella; Bruno P. Klaholz

doi:10.1093/nar/gkt1404

Purification, characterization and crystallization of the human 80S ribosome

Heena Khatter, Alexander G. Myasnikov, Leslie Mastio, Isabelle M.L. Billas, Catherine Birck, Stefano Stella, Bruno P. Klaholz^*

^*Corresponding author for this work

Protein Structure and Function Program

27 Citations (Scopus)

Abstract

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.

Original language	English
Journal	Nucleic Acids Research
Volume	42
Issue number	6
ISSN	0305-1048
DOIs	https://doi.org/10.1093/nar/gkt1404
Publication status	Published - 1 Jan 2014

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title = "Purification, characterization and crystallization of the human 80S ribosome",

abstract = "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 26{\AA} resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future highresolution work.",

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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.

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AN - SCOPUS:84898979617

SN - 0305-1048

VL - 42

JO - Nucleic Acids Research

JF - Nucleic Acids Research

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Purification, characterization and crystallization of the human 80S ribosome

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