Erik R. Farquhar

Packaging of viral genomes into progeny virions is a critical step in the viral life cycle. Coronaviruses such as SARS-CoV-2 possess unusually large RNA genomes (~30 kb), yet the mechanism by which these genomes are selectively condensed and incorporated into virions remains poorly understood. Here, we demonstrate that the SARS-CoV-2 genome contains multiple dispersed RNA structures, termed packaging signals (PSs), which cooperate with a dominant central PS to direct genomic RNA incorporation into infectious particles. We identify the dominant PS within the coding region of the nsp15 gene, downstream of a packaging signal previously described in Embecoviruses. Using virus-like particles (VLPs), we investigate its role in virion assembly and selective genomic RNA packaging, and show that it promotes the formation of ribonucleoprotein (RNP) complexes with the viral nucleocapsid protein (N), as revealed by mass photometry. Notably, we uncover a unique N-induced conformational rearrangement of the PS RNA, from an extended structure to a double stem-loop architecture. This dominant PS acts together with a nearby stem-loop element to assemble a higher-order RNP complex containing 12 N-protein dimers. Using a SARS-CoV-2 replicon system, we further demonstrate functional cooperativity between the dominant PS, its proximal partner stem-loop, and additional packaging elements located approximately 10 kb upstream. Collectively, our findings support a highly dynamic and cooperative mechanism of SARS-CoV-2 genome packaging that relies on multiple dispersed packaging signals organized around a dominant central PS. These insights provide a mechanistic framework for understanding coronavirus genome packaging and reveal new opportunities for antiviral intervention through disruption of this process. They also have important implications for vaccine development and may enable the design of membrane-based vector systems capable of efficiently delivering large nucleic acid cargoes, expanding the potential of bionanotechnology and genetic medicine.

Protein oxidative footprinting, using hydroxyl radical labeling detected by bottom-up proteomics, has progressed from an emerging method to a widely used approach in structural biology. Hydroxyl radicals generated from hydrogen peroxide (via photolysis, Fenton chemistry or electrochemistry) or directly from water (via X-rays, plasma or gamma rays) irreversibly encode structural information within protein side chains, which is read out using standard liquid chromatography-mass spectrometry workflows. Quantitative changes in labeling report on solvent accessibility and reveal effects of protein-protein interactions, ligand binding, protein folding, conformational changes or applied stress. Comparing labeling patterns between states provides detailed maps of structural changes and interaction sites. Over the past decade, oxidative footprinting has proven valuable as a solution-phase and in-cell method for protein structure analysis. This Perspective summarizes best practices for experimental design, sample processing, data analysis, interpretation and integration with orthogonal data, offering a consensus framework to guide application of oxidative footprinting in academic and biopharmaceutical research.

The ClearXCam detector is a new video rate imaging in-beam monitor based on a diamond sensor. Imaging with 2304 effective pixels is achieved by sequentially biasing one of 48 metal stripes on one side of a diamond sensor, while reading out the current from 48 stripes on the other side of the sensor. This system was characterized for real-time X-ray beam diagnostics at synchrotron beamline 17-BM at the National Synchrotron Light Source II. Significant results include: detection of in-beam structure via the imaging mode, beam focusing in one minute with real-time imaging feedback during a focusing event, linearity over five orders of magnitude, validation of a fast mode operating at 100 Hz, and sub-micron beam-positioning resolution. The system is now available commercially.

Summary: The early steps of eukaryotic large ribosomal subunit assembly remain poorly understood due to the structural flexibility of pre-60S intermediates, whose rRNA is extensively modified by small nucleolar RNPs (snoRNPs). Some snoRNPs, however, lack any modification function and instead scaffold ribosome assembly through largely unknown mechanisms. Here, we show that the H/ACA snoRNP snR37 integrates both modifying and scaffolding roles. Biochemical and structural analyses reveal a canonical H/ACA core that pseudouridylates a conserved uridine in the A site of the peptidyl transferase center, the catalytic heart of the 60S subunit. Additional RNA helices recruit non-core proteins, the Upa1-Upa2 heterodimer and Rbp95, which mediate stable snR37 association with pre-60S complexes. These proteins cooperate with the Npa1 rRNA chaperone complex to link four rRNA domains, thereby structurally organizing early pre-60S intermediates and promoting proper formation of the PTC. This dual organization establishes a paradigm for snoRNPs combining rRNA modification and scaffolding functions.

Shwachman‐Diamond syndrome (SDS) is a rare genetic disorder characterized by pancreatic insufficiency and neutropenia. While most cases are linked to mutations in the SBDS gene, some involve mutations in the GTPase EFL1. This protein works with SBDS to release the anti‐association factor eIF6 from the 60S ribosomal subunit during ribosome biogenesis. The pathogenic EFL1 R1095Q mutant (R1086Q in yeast) exhibits altered guanine nucleotide recognition and impaired eIF6 release, prompting an investigation into its structural consequences. Using the yeast Efl1 orthologue in X‐ray hydroxyl radical footprinting experiments, we tracked changes in solvent accessibility caused by the mutation. Although the mutation is situated in domain IV, widespread conformational changes were observed across the protein, particularly in domain I, suggesting a long‐range intramolecular communication. Strikingly, the growth defect caused by the pathogenic mutation was rescued by a second mutation located in a allosteric pathway that spans from the nucleotide‐binding pocket to domain IV. This compensatory mutation restored proper nuclear localization of eIF6 (Tif6 in yeast). These findings reveal that the R1086Q mutation disrupts a structural communication network within Efl1, impairing the conformational dynamics required for its activity. The loss of this coordination likely underlies the ribosome maturation defects observed in SDS cases linked to EFL1, offering new insights into the molecular basis of the disease.

The structural biology program at the National Synchrotron Light Source II presents a coordinated set of instruments, software and research opportunities for the interested user. We describe in some detail the research capabilities enabled by the Center for BioMolecular Structure. The evolution of the resources is described in detail, considering three major themes: automation, micro-focusing and computation prediction.

Protein oxidative ‘footprinting’— where obtaining structural information of proteins using hydroxyl radical labeling is detected with bottom-up proteomics mass spectrometry analysis—has evolved from an emerging technology to an approach applied to a range of structural biology and biophysics questions. Whether the hydroxyl radicals are generated from hydrogen peroxide (via photolysis, Fenton chemistry, or electrochemistry) or directly from water (through X-ray irradiation, plasma, or gamma rays), the resulting structural information is irreversibly encoded within the protein side chains and can be detected and quantitated with up to single-residue resolution, using a commonly applied set of LC-MS data reduction and analysis protocols. Quantitative measurements of changes in the oxidative labeling of amino acid side chain reflect alterations in solvent accessibility, typically resulting from protein-protein interactions, ligand binding, protein folding/conformational changes, or applied stress. Measuring changes in oxidative labeling thus provides a detailed map of structural changes and interaction sites across the system under study. Oxidative footprinting technologies over the past decade have demonstrated their utility as a mapping method for protein structure analysis in solution and within living cells. The goal of this article is to harmonize and disseminate best practices for experimental design, data collection, data interpretation, and data presentation. This perspective intends to facilitate the application of this insightful approach to new biological systems and experimental applications. The information presented in this article coalesces best practices independently developed by teams who have decades of experience in advancing protein oxidative footprinting. We provide a consensus viewpoint on the use of this technology and the different methodologies for structural elucidation in both academic research and in biopharmaceutical drug discovery and development.

Hydroxyl radical-based protein footprinting (HRPF) coupled with mass spectrometry is a valuable medium-resolution technique in structural biology, facilitating the assessment of protein structure and molecular-level interactions in solution conditions. In HRPF with X-rays (XFP), hydroxyl radicals generated by water radiolysis covalently label multiple amino acid (AA) side chains. However, HRPF technologies face challenges in achieving their full potential due to the broad (>10³) dynamic range of AA reactivity with ^•OH and difficulty in detecting slightly modified residues, most notably in peptides with highly reactive residues like methionine, or where all residues have low ^•OH reactivities. To overcome this limitation, we developed a multiplex labeling chemistry that utilizes both CF₃ radicals (^•CF₃) produced from a trifluoromethylation (TFM) reagent and OH radicals (^•OH), under controlled and optimized radiolysis doses generated by X-rays. We optimized the dual ^•CF₃/^•OH chemistry using model peptides and proteins, thereby extending the existing ^•OH labeling platform to incorporate simultaneous ^•CF₃ labeling. We labeled >50% of the protein sequence and >80% of protein solvent-accessible AAs via multiplex TFM labeling resulting in high-resolution footprinting, primarily by enhancing the labeling of AAs with low ^•OH reactivity via the ^•CF₃ channel, while labeling moderate and highly ^•OH-reactive AAs in both ^•CF₃ and ^•OH channels. Moreover, the low reactivity of methionine with ^•CF₃ enabled the detection and quantification of additional AAs labeled by ^•CF₃ within methionine-containing peptides. Finally, we found that the solvent accessibility of protein AAs directly correlated with ^•CF₃ labeling, demonstrating that multiplex TFM labeling enables a high-resolution assessment of molecular interactions for enhanced HRPF.