Ribosome Crystals Research Articles

Crystal structure of eukaryotic ribosome and its complexes with inhibitors.

A high-resolution structure of the eukaryotic ribosome has been determined and has led to increased interest in studying protein biosynthesis and regulation of biosynthesis in cells. The functional complexes of the ribosome crystals obtained from bacteria and yeast have permitted researchers to identify the precise residue positions in different states of ribosome function. This knowledge, together with electron microscopy studies, enhances our understanding of how basic ribosome processes, including mRNA decoding, peptide bond formation, mRNA, and tRNA translocation and cotranslational transport of the nascent peptide, are regulated. In this review, we discuss the crystal structure of the entire 80S ribosome from yeast, which reveals its eukaryotic-specific features, and application of X-ray crystallography of the 80S ribosome for investigation of the binding mode for distinct compounds known to inhibit or modulate the protein-translation function of the ribosome. We also refer to a challenging aspect of the structural study of ribosomes, from higher eukaryotes, where the structures of major distinctive features of higher eukaryote ribosome-the high-eukaryote-specific long ribosomal RNA segments (about 1MDa)-remain unresolved. Presently, the structures of the major part of these high-eukaryotic expansion ribosomal RNA segments still remain unresolved.This article is part of the themed issue 'Perspectives on the ribosome'.

Acoustic vibrations contribute to the diffuse scatter produced by ribosome crystals.

The diffuse scattering pattern produced by frozen crystals of the 70S ribosome from Thermus thermophilus is as highly structured as it would be if it resulted entirely from domain-scale motions within these particles. However, the qualitative properties of the scattering pattern suggest that acoustic displacements of the crystal lattice make a major contribution to it.

Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals.

In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.

Brave new RNA world.

Brave new RNA world.

Back to the future of RNA structure.

Back to the future of RNA structure.

Temperature-dependent radiation sensitivity and order of 70S ribosome crystals.

All evidence to date indicates that at T = 100 K all protein crystals exhibit comparable sensitivity to X-ray damage when quantified using global metrics such as change in scaling B factor or integrated intensity versus dose. This is consistent with observations in cryo-electron microscopy, and results because nearly all diffusive motions of protein and solvent, including motions induced by radiation damage, are frozen out. But how do the sensitivities of different proteins compare at room temperature, where radiation-induced radicals are free to diffuse and protein and lattice structures are free to relax in response to local damage? It might be expected that a large complex with extensive conformational degrees of freedom would be more radiation sensitive than a small, compact globular protein. As a test case, the radiation sensitivity of 70S ribosome crystals has been examined. At T = 100 and 300 K, the half doses are 64 MGy (at 3 Å resolution) and 150 kGy (at 5 Å resolution), respectively. The maximum tolerable dose in a crystallography experiment depends upon the initial or desired resolution. When differences in initial data-set resolution are accounted for, the former half dose is roughly consistent with that for model proteins, and the 100/300 K half-dose ratio is roughly a factor of ten larger. 70S ribosome crystals exhibit substantially increased resolution at 100 K relative to 300 K owing to cooling-induced ordering and not to reduced radiation sensitivity and slower radiation damage.

Structural basis of bacterial protein translation elongation

Recently, better understanding of ribosome function has been achieved through structural determination. These results provided a wealth of information on how genetic codes are precisely translated into proteins that control life, as well as established the platform to put ribosome as one of the richest validated targets for antibacterial drug discovery. We discovered a new ribosome crystal form obtained from ribosome lacking subunit L9, which allows crystallization of the ribosome in the presence of elongation factors Tu and G (EF-Tu/EF-G), trapped by antibiotics kirromycin and fusidic acid, respectively. These structures for the first time at atomic level offer insight into the molecular mechanism of protein elongation, which lies at the heart of translation. In each elongation cycle, EF-G facilitates the movement of tRNA-mRNA by one codon, which is coupled to the ratchet-like rotation of the ribosome complex and is triggered by EF-G-mediated GTP hydrolysis. The structure of pre-translocational ribosome bound to EF-G trapped with a GTP analogue, sheds light on how the positioning of the catalytic residue His87 into the active site is coupled to the hydrophobic gate opening which involves the sarcin-ricin loop (SRL) and domain III of EF-G. These results provide the structural basis for the GTPase activation of EF-G. After translocation, a cognate deacylated tRNA can only move together with the codon into the ribosomal E site. We also determined the structure of a cognate tRNA bound to the ribosomal E site, representing an authentic ribosome elongation complex. Quality control in protein translation by bacterial toxin has been associated with ribosome stalling and rescue system that is critical for cell to adapt environmental stress. Finally, I will introduce our structural progress on bacterial toxin bound to ribosome towards understanding of a classical acid-base catalysis mechanism.

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.

Serial femtosecond X-ray diffraction of 30S ribosomal subunit microcrystals in liquid suspension at ambient temperature using an X-ray free-electron laser.

High-resolution ribosome structures determined by X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. Here, the application of serial femtosecond X-ray crystallography (SFX) using an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature is described. 30S ribosomal subunit microcrystals diffracted to beyond 6 Å resolution, demonstrating the feasibility of using SFX for ribosome structural studies. The ability to collect diffraction data at near-physiological temperatures promises to provide fundamental insights into the structural dynamics of the ribosome and its functional complexes.

Ribosome engineering to promote new crystal forms.

Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors.

X-Ray Crystallography Refinement as Ewald Intended: From Drug Design to Ribosome Crystals

Precise protein-ligand complexes determined via X-ray crystallography experiments are critical for rational drug design. Here we demonstrate both improved statistical precision and greater chemical information content for a range of systems beginning with the trypsin-benzamidine complex shown below and scaling up to ribosome refinements. Keys to our method include use of the AMOEBA polarizable atomic multipole force field combined with a space group particle mesh Ewald (SG-PME) electrostatics algorithm that exploits space group symmetry to increase speed and reduce memory requirements. At high resolution (better than 1.0 A), we achieve lower R-free statistics by using the AMOEBA electron distribution within the X-ray scattering model. At all resolutions, rigorous treatment of polarizable atomic multipole electrostatics via SG-PME improves molecular conformation and orients the water hydrogen bonding network.View Large Image | View Hi-Res Image | Download PowerPoint Slide

A systematic study of 50S ribosomal subunit purification enabling robust crystallization

A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.9 A resolution and that were large enough to yield a complete data set from a single crystal. To our knowledge, this is the most systematic study of this challenging area so far undertaken. Ribosome crystallization is a complex multi-factorial problem and although a clear correlation of crystallization with subunit properties was not obtained, the search for key factors that potentiate crystallization has been greatly narrowed and promising areas for further inquiry are suggested.

Large facilities and the evolving ribosome, the cellular machine for genetic-code translation

Well-focused X-ray beams, generated by advanced synchrotron radiation facilities, yielded high-resolution diffraction data from crystals of ribosomes, the cellular nano-machines that translate the genetic code into proteins. These structures revealed the decoding mechanism, localized the mRNA path and the positions of the tRNA molecules in the ribosome and illuminated the interactions of the ribosome with initiation, release and recycling factors. They also showed that the ribosome is a ribozyme whose active site is situated within a universal symmetrical region that is embedded in the otherwise asymmetric ribosome structure. As this highly conserved region provides the machinery required for peptide bond formation and for ribosome polymerase activity, it may be the remnant of the proto-ribosome, a dimeric pre-biotic machine that formed peptide bonds and non-coded polypeptide chains. Synchrotron radiation also enabled the determination of structures of complexes of ribosomes with antibiotics targeting them, which revealed the principles allowing for their clinical use, revealed resistance mechanisms and showed the bases for discriminating pathogens from hosts, hence providing valuable structural information for antibiotics improvement.

Arrangement of 3′-terminus of tRNA on the human ribosome as revealed from cross-linking data

This study is directed towards an important problem concerning the organization of the peptidyl transferase center (PTC) on the mammalian ribosome that cannot be studied by X-ray analysis since crystals of 80S ribosomes are still unavailable. Here, we investigated the arrangement of the 3′-end of tRNA in the 80S ribosomal A and P sites using a tRNA Asp analogue that bears a 4-thiouridine (s 4U) attached to the 3′-terminal adenosine. It was shown that an additional nucleotide s 4U77 on the 3′-end does not impede codon-dependent binding of the tRNA to the A and P sites of 80S ribosome. Mild UV-irradiation of the ribosomal complexes containing a short appropriately designed mRNA and the tRNA analogue resulted in cross-linking of the analogue exclusively to 28S rRNA. The cross-linking site was detected in the 4302–4540 fragment of the 28S rRNA which belongs to the highly conserved domain V that in prokaryotic ribosomes is involved in the formation of the PTC. Nucleotides cross-linked to the tRNA analogue were determined by means of reverse transcription. A comparison of the results obtained with a dynamic model of mutual arrangement of s 4U77 of the A site tRNA and nucleotides of 23S rRNA built on the basis of an atomic model for the prokaryotic PTC led to the conclusion that environments of the tRNA 3′-terminus in prokaryotic and eukaryotic ribosomes share a significant extent of similarity, although pronounced differences are also detectable.

Structural Insights into Ribosome Recycling Factor Interactions with the 70S Ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.

Structure of the Base of the L7/L12 Stalk of the Haloarcula marismortui Large Ribosomal Subunit: Analysis of L11 Movements

Initiation factors, elongation factors, and release factors all interact with the L7/L12 stalk of the large ribosomal subunit during their respective GTP-dependent cycles on the ribosome. Electron density corresponding to the stalk is not present in previous crystal structures of either 50 S subunits or 70 S ribosomes. We have now discovered conditions that result in a more ordered factor-binding center in the Haloarcula marismortui (H.ma) large ribosomal subunit crystals and consequently allows the visualization of the full-length L11, the N-terminal domain (NTD) of L10 and helices 43 and 44 of 23 S rRNA. The resulting model is currently the most complete reported structure of a L7/L12 stalk in the context of a ribosome. This region contains a series of intermolecular interfaces that are smaller than those typically seen in other ribonucleoprotein interactions within the 50 S subunit. Comparisons of the L11 NTD position between the current structure, which is has an NTD splayed out with respect to previous structures, and other structures of ribosomes in different functional states demonstrates a dynamic range of L11 NTD movements. We propose that the L11 NTD moves through three different relative positions during the translational cycle: apo-ribosome, factor-bound pre-GTP hydrolysis and post-GTP hydrolysis. These positions outline a pathway for L11 NTD movements that are dependent on the specific nucleotide state of the bound ligand. These three states are represented by the orientations of the L11 NTD relative to the ribosome and suggest that L11 may play a more specialized role in the factor binding cycle than previously appreciated.

Reproducible growth of well diffracting ribosomal crystals

The crystallization of ribosomal particles is associated with extraordinary challenging demands. This originates mainly from the ribosome's natural tendency to deteriorate and from its multi-conformational heterogeneity, both of which stem from its functional flexibility. To increase the level of homogeneity of ribosomal preparations, systematic searches for conditions yielding populations of fully defined chemical compositions were employed and the variables essential for high functional activity were analyzed and optimized. These include temperature, cell-growth duration and media, the cell-harvesting stage, ribosomal purification and storage. The functional state that is most suitable to yield quality crystals was identified as that of the polysome and it was found that this fraction reproducibly yielded crystals of superior properties.

Variable and conserved elements of human ribosomes surrounding the mRNA at the decoding and upstream sites.

This study is centred upon an important biological problem concerning the structural organization of mammalian ribosomes that cannot be studied by X-ray analysis because 80S ribosome crystals are still unavailable. Here, positioning of the mRNA on 80S ribosomes was studied using mRNA analogues containing the perfluorophenylazide cross-linker on either the guanosine or an uridine residue. The modified nucleotides were directed to positions from -9 to +6 with respect to the first nucleotide of the P site bound codon by a tRNA cognate to the triplet targeted to the P site. Upon mild UV-irradiation, the modified nucleotides at positions +4 to +6 cross-linked to protein S15 and 18S rRNA nucleotides A1823-A1825. In addition, modified guanosines in positions +5 and +6 also cross-linked to G626, and in position +1 to G1702. Cross-linking from the upstream positions was mainly to protein S26 that has no prokaryotic homologues. These findings indicate that the tail of mammalian S15 comes closer to the decoding site than that of its prokaryotic homologue S19, and that the environments of the upstream part of mRNA on 80S and 70S ribosomes differ. On the other hand, the results confirm the widely accepted idea regarding the conserved nature of the decoding site of the small subunit rRNA.

Altering the morphology of crystals of a macromolecule without changing the unit cell (A case study of ribosome crystals)

Crystals of different morphology (needle-, rod-tetragonal- and wedge-shaped) were grown from the same solution of ribosome and in the same drop using 10–15% 2-methyl-2,4-pentanediol as a precipitant. The crystals appeared in 5–48 h. Observations of the kinetics of crystal appearance lead to the conclusion that higher supersaturation of ribosome solution produces needle-shaped crystals, while the tetragonal-rod- and the wedge-shaped crystals grow at lower supersaturation. The ribosome packing in the crystals was characterized by electron microscopy (EM). An analysis of EM ultrathin sections indicates that the unit cells, with a high degree of probability, are the same in the needle-shaped and other morphology crystals. The data obtained suggest that adjusting supersaturation of the crystallization solution may convert needle-shaped to more useful morphologies.

Metal Compounds as Tools for the Construction and the Interpretation of Medium-Resolution Maps of Ribosomal Particles

Procedures were developed exploiting organometallic clusters and coordination compounds in combination with heavy metal salts for derivatization of ribosomal crystals. These enabled the construction of multiple isomorphous replacement (MIR) and multiple isomorphous replacement combined with anomalous scattering medium-resolution electron density maps for the ribosomal particles that yield the crystals diffracting to the highest resolution, 3 Å, of the large subunit from Haloarcula marismortui and the small subunit from Thermus thermophilus. The first steps in the interpretation of the 7.3-Å MIR map of the small subunit were made with the aid of a tetrairidium cluster that was covalently attached to exposed sulfhydryls on the particle's surface prior to crystallization. The positions of these sulfhydryls were localized in difference Fourier maps that were constructed with the MIR phases.