The structure of the human 80S ribosome at 1.9 Å resolution reveals the molecular role of chemical modifications and ions in RNA.

The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. Here we present the 1.9 Å resolution cryo electron microscopy structure of the 80S human ribosome resolving numerous new ribosomal RNA modifications and functionally important ions such as Zn, K and Mg, including their associated individual water molecules.

Focused classifications and refinements in high-resolution single particle cryo-EM analysis.

Recent advances in cryo electron microscopy (cryo-EM) and image processing provide new opportunities to analyse drug targets at high resolution. However, structural heterogeneity limits resolution in many practical cases, hence restricting the level at which structural details can be analysed and drug design be performed. As structural disorder is not spread throughout the entire structure of a given macromolecular complex but instead is found in certain regions that move with respect to others and covering molecular scales from domain conformational changes up to the level of side chain conformations in ligand binding pockets, it is possible to focus the attention on those regions and the associated relative movements.

Asymmetric dimerization in a transcription factor superfamily is promoted by allosteric interactions with DNA.

Transcription factors, such as nuclear receptors achieve precise transcriptional regulation by means of a tight and reciprocal communication with DNA, where cooperativity gained by receptor dimerization is added to binding site sequence specificity to expand the range of DNA target gene sequences. To unravel the evolutionary steps in the emergence of DNA selection by steroid receptors (SRs) from monomeric to dimeric palindromic binding sites, we carried out crystallographic, biophysical and phylogenetic studies, focusing on the estrogen-related receptors (ERRs, NR3B) that represent closest relatives of SRs. Our results, showing the structure of the ERR DNA-binding domain bound to a palindromic response element (RE), unveil the molecular mechanisms of ERR dimerization which are imprinted in the protein itself with DNA acting as an allosteric driver by allowing the formation of a novel extended asymmetric dimerization region (KR-box).

High-resolution cryo-EM performance comparison of two latest-generation cryo electron microscopes on the human ribosome.

Recent technological advances in cryo electron microscopy (cryo-EM) have led to new opportunities in the structural biology field. Here we benchmark the performance of two 300 kV latest-generation cryo electron microscopes, Titan Krios G4 from Thermofisher Scientific and CRYO ARM 300 from Jeol, with regards to achieving high resolution single particle reconstructions on a real case sample. We compare potentially limiting factors such as drift rates, astigmatism & coma aberrations and performance during image processing and show that both microscopes, while comprising rather different technical setups & parameter settings and equipped with different types of energy filters & cameras, achieve a resolution of around 2 Å on the human ribosome, a non-symmetric object which constitutes a key drug target.

A novel nuclear receptor subfamily enlightens the origin of heterodimerization.

Background: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species.

Visualizing the Role of 2'-OH rRNA Methylations in the Human Ribosome Structure.

Chemical modifications of RNA have recently gained new attention in biological sciences. They occur notably on messenger RNA (mRNA) and ribosomal RNA (rRNA) and are important for various cellular functions, but their molecular mechanism of action is yet to be understood in detail. Ribosomes are large ribonucleoprotein assemblies, which synthesize proteins in all organisms.

Volta phase plate data collection facilitates image processing and cryo-EM structure determination.

A current bottleneck in structure determination of macromolecular complexes by cryo electron microscopy (cryo-EM) is the large amount of data needed to obtain high-resolution 3D reconstructions, including through sorting into different conformations and compositions with advanced image processing. Additionally, it may be difficult to visualize small ligands that bind in sub-stoichiometric levels. Volta phase plates (VPP) introduce a phase shift in the contrast transfer and drastically increase the contrast of the recorded low-dose cryo-EM images while preserving high frequency information.

Visualization of chemical modifications in the human 80S ribosome structure.

Chemical modifications of human ribosomal RNA (rRNA) are introduced during biogenesis and have been implicated in the dysregulation of protein synthesis, as is found in cancer and other diseases. However, their role in this phenomenon is unknown. Here we visualize more than 130 individual rRNA modifications in the three-dimensional structure of the human ribosome, explaining their structural and functional roles.

Focused classification and refinement in high-resolution cryo-EM structural analysis of ribosome complexes.

Cryo electron microscopy (cryo-EM) historically has had a strong impact on the structural and mechanistic analysis of protein synthesis by the prokaryotic and eukaryotic ribosomes. Vice versa, studying ribosomes has helped moving forwards many methodological aspects in single particle cryo-EM, at the level of automated data collection and image processing including advanced techniques for particle sorting to address structural and compositional heterogeneity. Here we review some of the latest ribosome structures, where cryo-EM allowed gaining unprecedented insights based on 3D structure sorting with focused classification and refinement methods helping to reach local resolution levels better than 3Å.

Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor.

Most nuclear receptors (NRs) bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs) involves specific protein-DNA and protein-protein interactions. The estrogen-related receptor (ERR) belongs to the steroid hormone nuclear receptor (SHR) family and shares strong similarity in its DNA-binding domain (DBD) with that of the estrogen receptor (ER).

The integrative role of cryo electron microscopy in molecular and cellular structural biology.

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo-EM) is now heading off at unprecedented speed towards high-resolution analysis of biological objects of various sizes. This 'revolution in resolution' is happening largely thanks to new developments of new-generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo-EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell.

Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor.

Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl(fMet)-tRNA(fMet) positioning and efficient transpeptidation are affected.

Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms.

Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated.

Crystal packing modifies ligand binding affinity: the case of aldose reductase.

The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed.

Neutron macromolecular crystallography with LADI-III.

At the Institut Laue-Langevin, a new neutron Laue diffractometer LADI-III has been fully operational since March 2007. LADI-III is dedicated to neutron macromolecular crystallography at medium to high resolution (2.5-1.

X-ray-induced deterioration of disulfide bridges at atomic resolution.

Overall and site-specific X-ray-induced damage to porcine pancreatic elastase was studied at atomic resolution at temperatures of 100 and 15 K. The experiments confirmed that irradiation causes small movements of protein domains and bound water molecules in protein crystals. These structural changes occur not only at 100 K but also at temperatures as low as 15 K.

Incorporation of methyl-protonated valine and leucine residues into deuterated ocean pout type III antifreeze protein: expression, crystallization and preliminary neutron diffraction studies.

Antifreeze proteins (AFPs) are found in different species from polar, alpine and subarctic regions, where they serve to inhibit ice-crystal growth by adsorption to ice surfaces. Recombinant North Atlantic ocean pout (Macrozoarces americanus) AFP has been used as a model protein to develop protocols for amino-acid-specific hydrogen reverse-labelling of methyl groups in leucine and valine residues using Escherichia coli high-density cell cultures supplemented with the amino-acid precursor alpha-ketoisovalerate. Here, the successful methyl protonation (methyl reverse-labelling) of leucine and valine residues in AFP is reported.

X-ray-radiation-induced cooperative atomic movements in protein.

X-rays interact with biological matter and cause damage. Proteins and other macromolecules are damaged primarily by ionizing X-ray photons and secondarily by reactive radiolytic chemical species. In particular, protein molecules are damaged during X-ray diffraction experiments with protein crystals, which is, in many cases, a serious hindrance to structure solution.

Unusual binding mode of the 2S4R stereoisomer of the potent aldose reductase cyclic imide inhibitor fidarestat (2S4S) in the 15 K crystal structure of the ternary complex refined at 0.78 A resolution: implications for the inhibition mechanism.

The structure of human aldose reductase in complex with the 2 S4 R stereoisomer of the potent inhibitor Fidarestat ((2 S,4 S)-6-fluoro-2',5'-dioxospiro-[chroman-4,4'-imidazoline]-2-carboxamide) was determined at 15 K and a resolution of 0.78 A. The structure of the complex provides experimental evidence for the inhibition mechanism in which Fidarestat is initially bound neutral and then becomes negatively charged by donating the proton at the 1'-position nitrogen of the cyclic imide ring to the N2 atom of the catalytic His110.

Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase.

We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 A, 100K; 0.80 A, 15K; 1.

The atomic resolution structure of human aldose reductase reveals that rearrangement of a bound ligand allows the opening of the safety-belt loop.

The crystal structure of human aldose reductase in complex with citrate has been determined to a resolution of 0.82 A. The difference electron density for H atoms unequivocally shows that the cofactor is in the oxidized state corresponding to the situation after the catalytic event has occurred.

Ultrahigh-resolution study of protein atomic displacement parameters at cryotemperatures obtained with a helium cryostat.

Two X-ray data sets for a complex of human aldose reductase (h-AR) with the inhibitor IDD 594 and the cofactor NADP(+) were collected from two different parts of the same crystal to a resolution of 0.81 A at 15 and 60 K using cold helium gas as cryogen. The contribution of temperature to the atomic B values was estimated by comparison of the independently refined models.

Factorizing selectivity determinants of inhibitor binding toward aldose and aldehyde reductases: structural and thermodynamic properties of the aldose reductase mutant Leu300Pro-fidarestat complex.

Structure of the Leu300Pro mutant of human aldose reductase (ALR2) in complex with the inhibitor fidarestat is determined. Comparison with the hALR2-fidarestat complex and the porcine aldehyde reductase (ALR1)-fidarestat complex indicates that the hydrogen bond between the Leu300 amino group of the wild-type and the exocyclic amide group of the inhibitor is the key determinant for the specificity of fidarestat for ALR2 over ALR1. Thermodynamic data also suggest an enthalpic contribution as the predominant difference in the binding energy between the aldose reductase mutant and the wild-type.