MODOMICS: a database of RNA modifications and related information. 2023 update.

The MODOMICS database was updated with recent data and now includes new data types related to RNA modifications. Changes to the database include an expanded modification catalog, encompassing both natural and synthetic residues identified in RNA structures. This addition aids in representing RNA sequences from the RCSB PDB database more effectively. To manage the increased number of modifications, adjustments to the nomenclature system were made. Updates in the RNA sequences section include the addition of new sequences and the reintroduction of sequence alignments for tRNAs and rRNAs. The protein section was updated and connected to structures from the RCSB PDB database and predictions by AlphaFold. MODOMICS now includes a data annotation system, with 'Evidence' and 'Estimated Reliability' features, offering clarity on data support and accuracy. This system is open to all MODOMICS entries, enhancing the accuracy of RNA modification data representation. MODOMICS is available at https://iimcb.genesilico.pl/modomics/.

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. Here we show how the usage of focused classifications and refinements provide insights into global conformational arrangements, exemplified on the human ribosome and on the cannabinoid G protein coupled receptor (GPCR), and how they can improve the local map resolution from an essentially disordered region to the 3-4 Å and finally to the 2 Å resolution range. A systematic analysis with variable spherical masks during focused refinement is presented showing that the choice of an optimal mask size helps refining to high resolution. This study covers several practical approaches on 4 examples illustrating how important mask size & shape and including neighbouring structural elements are for a focused analysis of a macromolecular complex. Such methods will be crucial for cryo-EM structure-based drug design of various medical targets and are applicable to single particle cryo-EM and electron tomography data.

Program VUE: analysing distributions of cryo-EM projections using uniform spherical grids.

Three-dimensional cryo electron microscopy reconstructions are obtained by extracting information from a large number of projections of the object. These projections correspond to different 'views' or 'orientations', i.e. directions in which these projections show the reconstructed object. Uneven distribution of these views and the presence of dominating preferred orientations may distort the reconstructed spatial images. This work describes the program VUE (views on uniform grids for cryo electron microscopy), designed to study such distributions. Its algorithms, based on uniform virtual grids on a sphere, allow an easy calculation and accurate quantitative analysis of the frequency distribution of the views. The key computational element is the Lambert azimuthal equal-area projection of a spherical uniform grid onto a disc. This projection keeps the surface area constant and represents the frequency distribution with no visual bias. Since it has multiple tunable parameters, the program is easily adaptable to individual needs, and to the features of a particular project or of the figure to be produced. It can help identify problems related to an uneven distribution of views. Optionally, it can modify the list of projections, distributing the views more uniformly. The program can also be used as a teaching tool.

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 Zn2+, K+ and Mg2+, including their associated individual water molecules. The 2'-O-methylation, pseudo-uridine and base modifications were confirmed by mass spectrometry, resulting in a complete investigation of the >230 sites, many of which could not be addressed previously. They choreograph key interactions within the RNA and at the interface with proteins, including at the ribosomal subunit interfaces of the fully assembled 80S ribosome. Uridine isomerization turns out to be a key mechanism for U-A base pair stabilization in RNA in general. The structural environment of chemical modifications and ions is primordial for the RNA architecture of the mature human ribosome, hence providing a structural framework to address their role in healthy states and in human diseases.