DOI: 10.1093/nar/gkt1404. Purification, characterization and crystallization of the human 80S ribosome

Purification, characterization and crystallization of the human 80S ribosome

10.1093/nar/gkt1404

Crossref journal-article

Oxford University Press (OUP)

Nucleic Acids Research (286)

Abstract

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 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.

Bibliography

Khatter, H., Myasnikov, A. G., Mastio, L., Billas, I. M. L., Birck, C., Stella, S., & Klaholz, B. P. (2014). Purification, characterization and crystallization of the human 80S ribosome. Nucleic Acids Research, 42(6), e49âe49.

Authors 7

Heena Khatter (first)
Alexander G. Myasnikov (additional)
Leslie Mastio (additional)
Isabelle M. L. Billas (additional)
Catherine Birck (additional)
Stefano Stella (additional)
Bruno P. Klaholz (additional)

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Dates

Type	When
Created	11 years, 7 months ago (Jan. 22, 2014, 8:35 p.m.)
Deposited	3 years, 1 month ago (Aug. 1, 2022, 9:41 a.m.)
Indexed	3 months, 1 week ago (May 27, 2025, 1:04 p.m.)
Issued	11 years, 7 months ago (Jan. 21, 2014)
Published	11 years, 7 months ago (Jan. 21, 2014)
Published Online	11 years, 7 months ago (Jan. 21, 2014)
Published Print	11 years, 5 months ago (April 1, 2014)

Funders 0

None

BibTeX

@article{Khatter_2014, title={Purification, characterization and crystallization of the human 80S ribosome}, volume={42}, ISSN={0305-1048}, url={http://dx.doi.org/10.1093/nar/gkt1404}, DOI={10.1093/nar/gkt1404}, number={6}, journal={Nucleic Acids Research}, publisher={Oxford University Press (OUP)}, author={Khatter, Heena and Myasnikov, Alexander G. and Mastio, Leslie and Billas, Isabelle M. L. and Birck, Catherine and Stella, Stefano and Klaholz, Bruno P.}, year={2014}, month=jan, pages={e49–e49} }

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