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 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.

Figure 1.

Biophysical characterization of human 80S ribosomes purified from HeLa cells. (A) 15–30% sucrose density gradient profiles for 80S, and dissociated 60S and 40S subunits depicting the separation of components based on density. (B) SEC-MALLS for 80S sample and (C) AUC results showing the homogeneity of 80S sample, which corresponds to the calculated molecular weight of 4.3 MDa.

Figure 2.

Cryo electron micrograph depicting the distribution of 80S particles at 59k magnification, collected using a Polara F-30 electron microscope. The ribosomes were isolated from Hela cells (A) under normal growth conditions (B) in stationary phase of growth (C) after glutamine starvation (D) after serum-starvation (inset shows the zoomed-in view for individual ribosomes). (E) The cryo-EM structure of empty human 80S and with (F) E-site tRNA; both isolated from HeLa cells; under high and low KCl concentration during Puromycin treatment, respectively; color code: 40S golden, 60S blue, tRNA red.

Figure 3.

(A) First human 80S ribosome crystals obtained in the capillaries of a crystal harp. (B) Crystals with plate morphology reproduced in sitting drops that diffracted up to 26 Å, most are seen on the edge and thus give the impression of rods. (C) 80S crystal mounted in a cryogenic loop, tested for X-ray diffraction at the PX-II beamline at SLS. (D) Agarose gel depicting the sample stability under crystallization conditions as monitored by the presence of 18S and 28S rRNA. Lane 1 shows control ribosomes stored at −80°C, lanes 2 and 3 show ribosomes incubated with or without precipitant, respectively, for 4 weeks at 4°C. (E) The diffraction pattern shows a full reciprocal lattice with cell parameters of approximately a = 406 Å, b = 785 Å, c = 977 Å, with resolution rings indicated at 23, 30 and 40 Å. The inset shows diffraction spots extending to 26 Å resolution.