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Cross-linking mass spectrometry studies protein interactions and structures but struggles with identifying crosslinked peptides. Here, the authors present Prosit-XL, a fragment intensity predictor that improves crosslinked peptide identification, boosting discovery of interactions and structures.

Cross-linking mass spectrometry (MS) can identify protein-protein interaction (PPI) networks but assessing the reliability of these data remains challenging. To address this issue, the authors develop and validate a method to determine the false-discovery rate of PPIs identified by cross-linking MS.

Predicting chromatographic retention times (RTs) has proven beneficial in proteomics but has not yet been achieved for crosslinked peptides. Here, the authors develop an RT prediction tool for crosslinked peptides and leverage predicted RTs to increase identifications in crosslinking mass spectrometry studies.

Quantitative cross-linking/mass spectrometry is used to discover protein structural changes (e.g., in protein interactions). The cross-linker bis(sulfosuccinimidyl)suberate is used for both label-free and isotope-labeled workflows in this protocol.

Elucidating the structure of protein complexes is key to understanding life at the molecular level. Here, the authors improve modelling performance on challenging targets by integrating experimental distance restraints from crosslinking mass spectrometry into AlphaFold-Multimer.

Human protein co-regulation map is derived from a large set of quantitative proteomics experiments.

The structure of the multiprotein Fanconi anaemia core complex, determined using cryo-electron microscopy and mass spectrometry, shows that the complex adopts an extended asymmetric structure and highlights the structural and functional asymmetry of the RING finger domains.

Using cryo-EM, the authors show that the mammalian CCR4–NOT complex specifically recognizes stalled translating ribosomes similar to the yeast complex, locks them in a translation-incompetent state and coordinates their ubiquitylation, highlighting its central role in linking translation to mRNA stability.

Cryo-electron microscopy and tomography structures of reconstituted and endogenous human mRNA ribonucleoprotein complexes bound to the transcription–export complex reveal how mRNAs are packaged and recognized for nuclear export.

Two evolutionarily distant SMC–kleisin complexes are shown to contain a bendable coiled-coil discontinuity near the middle of their arms, which permits a folded conformation with potential implications for DNA loading and translocation.

Limitations of Alphafold2 structure prediction are addressed by including experimentally determined distance constraints.

Kustatscher et al. describe ChEP, an improvement on the classic chromatin pellet method that enables users to take a 'snapshot' of chromatin, making possible the isolation and identification of the full range of chromatin-binding proteins.

RNA modifications, including 5-methylcytosines, are known regulators of RNA metabolism affecting protein-RNA binding. Here, the authors reveal the role of m⁵C in the biogenesis of the non-coding vault-derived small RNAs.

Lamin A is critical for nuclear architecture but its structure and assembly are not fully understood. Here, the authors use quantitative cross-linking mass spectrometry to map intra- and intermolecular interactions within lamin homomers, providing insights into the molecular basis for lamin’s mechanical properties.

In male mouse germline development, the precise DNA methylation of young, active transposons requires a two-step process in which SPIN1 and SPOCD1 mark young LINE1 elements before the piRNA pathway triggers DNA methylation.

The inhibitor of kB kinase (IKK) is a central regulator of NF-kB signalling. Here the authors identify a motif conserved in substrates of canonical and alternative NF-kB pathways which mediates docking to catalytic IKK dimers: they show that phosphorylation of the conserved tyrosine suppresses the docking interaction.

A 3.9-Å-resolution cryo-EM structure of the S. cerevisiae OCCM replicative helicase loading complex bound to DNA shows how ORC and Cdc6 recognize DNA origins, and reveals details of how the Mcm2–7 hexamer ring is loaded onto the DNA helix.

One of two papers in this issue that identifies enzymes capable of demethylating a tri-methyl group from Lys 9 of histone H3 — a mark required for the establishment of heterochromatin and previously considered to be stable. GASC1, a member of the JMJD2 enzyme family, can disrupt heterochromatin structure when overexpressed and may contribute to tumour development.

Two human JmjC-___domain-containing enzymes are shown to catalyse demethylation of histone H3K27me3, a histone modification mediated by polycomb proteins and associated with stem cell maintenance and differentiation

Centromere protein A (CENP-A) is a histone H3 variant that specifies centromere ___location. Their reduced height relative to canonical H3 nucleosomes suggested that CENP-A nucleosomes might be tetrameric, but new biophysical measurements of reconstituted nucleosomes show that CENP-A confers a reduction in height on octameric CENP-A nucleosomes.

How archaeal viruses perturb host transcription machinery is poorly understood. Here, the authors provide evidence that the archaeo-viral transcription factor ORF145/RIP targets host RNA polymerase, repressing its activity.

Here the authors determined several cryo-EM structures of the human Elongator complex, which modifies anticodons of tRNAs. The structural work is complemented by functional analyses to understand this clinically relevant cellular machine at the molecular level.

Using cryo-electron tomography, Dendooven et al. determined the structure of the native budding yeast γ-tubulin ring complex (γTuRC) capping spindle microtubules and showed that γTuRC adopts an active closed conformation to function as a perfect geometric template for microtubule nucleation.

The African trypanosome Trypanosoma brucei has been shown to form stress granules in vitro that might be repurposed to enable differentiation and facilitate parasite transmission. Here, Cayla et al. show that differentiation between slender and stumpy forms does involve membrane-less granules that are different from nutritional stress granules.

TFIID is an essential transcription factor complex that controls the expression of most protein-coding genes in eukaryotes. Here the authors identify and characterize a complex containing TAF2, TAF8 and TAF10, which assembles in the cytoplasm before integration into the nuclear holo–TFIID complex.

Newly identified protein SPOCD1 is crucial in de novo DNA methylation directed by PIWI proteins and piRNAs, helping to control DNA silencing in mouse male germline.

During nuclear surveillance in yeast and human cells, the RNA exosome functions together with the TRAMP complexes. Here, the authors defined the protein composition of the TRAMP complexes and identified specific RNA binding sites for the different TRAMP components.

The PIWI protein MIWI2 counteracts transposon activity by transcriptional silencing in the mammalian germline. Here, the authors show that TEX15 interacts with MIWI2 and is required for piRNA-directed methylation of transposable elements in male germ cells.

The cryo-electron microscopy structure of human thyroglobulin reveals that proximity, flexibility and solvent exposure are key characteristics of its hormonogenic tyrosine pairs, and provides a framework for understanding the formation of thyroid hormones.

Sijacki et al. show how phosphorylation of FANCI by the ATR DNA damage kinase primes the FANCD2–FANCI clamp for ubiquitination. Phosphorylation promotes closure of the clamp, exposing a lysine for ubiquitination to initiate DNA cross-link repair.

Cryo-electron tomography is used to reveal the structural dynamics and functional diversity of translating ribosomes in Mycoplasma pneumoniae, providing insight into the translation elongation cycle inside cells and how it is reshaped by antibiotics.

Here, the authors find that histone demethylase Epe1-mediated stress resistance is regulated by proteasome-dependent truncation, allowing heterochromatin to reprogram gene expression that confers antifungal resistance to some fission yeast cells in the population.

Tsr1 is an essential ribosome biogenesis factor that has known similarity to GTPases. Here, the authors report the Tsr1 crystal structure and show that it is similar to GTPases but that active site residues are not conserved; modelling of the structure into the pre-40S maps allows inferences on ribosomal maturation to be drawn.

To ensure genome stability, cells need to restrict DNA replication to once per cell cycle. Here the authors show that Cyclin F interacts with and targets the licensing factor CDC6 for degradation, preventing re-firing of replication origins.

Kinetochores must interact with both polymerizing (straight) and depolymerizing (curved) microtubules to ensure correct mitotic chromosome segregation. Abad et al. reveal how this flexibility is achieved through structural characterization of the interactions between microtubules and the kinetochore protein Ska1.

In this study, the authors show that MeCP2 interacts with the NCoR/SMRT co-repressor complex and that a discrete cluster of Rett syndrome–causing mutations in the C-terminal ___domain of MeCP2 disrupts this interaction, impairing transcriptional repression. Knock-in mice expressing one of these MeCP2 missense mutations exhibit severe motor phenotypes.

The phosphatidylinositol-3-phosphate (PI3P) is generated by the lipid kinase VPS34, in the context of VPS34 complex I on autophagosomes or complex II on endosomes. Biochemical and structural analyses provide insights into the mechanism of both VPS34 complexes recruitment to and activation on membranes by specific Rab GTPases.

The bacterial helicase-like transcription factor HelD interacts with the RNA polymerase (RNAP) and together with the RNAP δ subunit enhances RNAP cycling. Here, the authors present the cryo-EM structures of the monomeric and dimeric Bacillus subtilis RNAP-δ-HelD complexes and suggest a model for HelD/δ-mediated RNAP recycling and putative hibernation.

Cryo-EM structures of the S. cerevisiae condensin holo complex reveal that ATP binding triggers exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains, potentially leading to an interconversion of DNA-binding sites in the catalytic core of condensin that might form the basis of its DNA translocation and loop-extrusion activities.

Gain-of-function mutations altering the PWWP ___domain of DNMT3A are identified as a new cause of microcephalic dwarfism. These mutations abrogate DNMT3A binding to H3K36me2 and H3K36me3 and lead to aberrant DNA methylation of Polycomb-marked regions.

Oligomers of human αA-crystallin are characterized structurally via a hybrid approach, combining cryo-EM, cross-linking/mass spectrometry, NMR and modeling, providing insight into their dynamic behavior and heterogeneity and revealing that oxidized oligomers can also act as chaperones.

Structural analysis of the DNA crosslink repair factors FANCD2 and FANCI demonstrates that a complex of both factors adopts a closed conformation upon ubiquitination of FANCD2, trapping the complex on DNA.

Groth and colleagues identify proteins associated with newly synthesized DNA — isolated by nascent chromatin capture — as well as proteins associated with mature DNA, and find factors not previously linked to replication or nascent chromatin.