Conny Wing-Heng Yu

Structure integration with function, taxonomy and sequences (SIFTS) provides residue‐level mappings between UniProt Knowledgebase sequences and Protein Data Bank structures and has historically been generated through internal Protein Data Bank in Europe (PDBe) pipelines. Here, PDBe‐SIFTS is presented as a fully open‐source, locally deployable implementation of this mapping framework. The pipeline combines fast, scalable sequence search using MMseqs2, an improved bounded scoring scheme for ranking candidate mappings, and residue‐level mapping refinement based on backbone connectivity. PDBe‐SIFTS is distributed as a Python package with command‐line tools for (1) building a sequence search database, (2) identifying the best sequence‐structure match, (3) one‐to‐one mapping at the residue level, and (4) generating SIFTS annotations in PDBx/mmCIF format. Benchmarking on the complete Protein Data Bank archive showed that MMseqs2 reduced archive‐scale UniProtKB searches from hours with BLASTP to minutes, approximately 22–36 times faster, while curated mappings were recovered at top rank in 93.1% of cases. The remaining discrepancies mainly involved biologically ambiguous cases such as highly conserved proteins, chimeric constructs, or closely related orthologs. These results show that PDBe‐SIFTS enables fast mapping, improving structural coherence in residue‐level alignments while delivering the most up‐to‐date and accurate mappings, comparable to expert curation. Tool: https://github.com/PDBeurope/SIFTS . Quick start notebook with example: https://github.com/PDBeurope/SIFTS/tree/master/notebooks .

UniProt provides the scientific community with a comprehensive, high-quality and freely accessible resource of protein sequence and functional information. This webinar will guide you through ways in which students and researchers at all career stages may take a structured approach to access the data in the UniProtKB and Proteomes sections of the UniProt protein function database. We will discuss the types of protein data that can be accessed, how to navigate the UniProt website, and where UniProt’s data comes from. We will demonstrate how to: intuitively search the UniProt website; find the function of a protein; identify the protein components of a pathway; compare proteins features of interest; determine protein orthologs in other species; obtain data sets containing all the proteins expressed in an organism. By accessing protein sequence, structure and functional data through UniProt, researchers can discover new protein data, expand their biological knowledge of target protein families and pathways, and ultimately drive further scientific discoveries.

Structure Integration with Function, Taxonomy and Sequences (SIFTS) provides residue-level mappings between UniProt Knowledgebase sequences and Protein Data Bank structures and has historically been generated through internal Protein Data Bank in Europe (PDBe) pipelines. Here, PDBe-SIFTS is presented as a fully open-source, locally deployable implementation of this mapping framework. The pipeline combines fast, scalable sequence search using MMseqs2, an improved bounded scoring scheme for ranking candidate mappings, and residue-level mapping refinement based on backbone connectivity. PDBe-SIFTS is distributed as a Python package with command-line tools for 1) building a sequence search database, 2) identifying the best sequence-structure match, 3) one-to-one mapping at the residue level, and 4) generating SIFTS annotations in PDBx/mmCIF format. Benchmarking on the complete Protein Data Bank archive showed that MMseqs2 reduced archive-scale UniProtKB searches from hours with BLASTP to minutes, approximately 22-36 times faster, while curated mappings were recovered at top rank in 93.1% of cases. The remaining discrepancies mainly involved biologically ambiguous cases such as highly conserved proteins, chimeric constructs, or closely related orthologs. These results show that PDBe-SIFTS enables fast mapping, improving structural coherence in residue-level alignments while delivering the most up-to-date and accurate mappings, comparable to expert curation. Tool: https://github.com/PDBeurope/SIFTS Quick start notebook with example: https://github.com/PDBeurope/SIFTS/tree/master/notebooks Broader audience statement: Matching protein sequences to their three-dimensional structures, and mapping annotations across both, is essential for understanding protein function, interactions, and molecular mechanisms. This integrated view enables richer interpretation of biological data and underpins advances in drug discovery, disease research, and protein engineering. PDBe-SIFTS provides an open and functional framework for structure–sequence mapping, allowing researchers and databases to run, inspect, and extend these mappings locally, while benefiting from faster searches, transparent scoring, and structurally informed residue-level alignments. Graphical

With over 140,000 experimentally determined structures and more than 200 million predicted structural models mapped to its entries, UniProt is one of the world's most comprehensive structure-annotated protein databases. In this webinar, we will provide a walkthrough on how to access and explore structural data associated with a protein entry in UniProt. We will describe the various types of structural information captured in UniProt, from topological arrangements to residue-level annotations. We will demonstrate how to find all available structures for a protein and how to analyse individual components of multi-protein complexes. We will also introduce how this information can be retrieved via the UniProt Proteins API using basic Python.

During mitosis, unattached kinetochores trigger the spindle assembly checkpoint by promoting assembly of the mitotic checkpoint complex, a heterotetramer comprising Mad2, Cdc20, BubR1 and Bub3. Critical to this process is the kinetochore-mediated catalysis of an intrinsically slow conformational conversion of Mad2 from an open (O-Mad2) inactive state to a closed (C-Mad2) active state bound to Cdc20. These Mad2 conformational changes involve substantial remodelling of the N-terminal β1 strand and C-terminal β7/β8 hairpin. In vitro , the Mad2- interaction motif (MIM) of Cdc20 (Cdc20 ^MIM ) triggers rapid conversion of O- to C-Mad2, effectively removing the kinetic barrier for MCC assembly. How Cdc20 ^MIM directly induces Mad2 conversion remains unclear. In this study we demonstrate that the Cdc20 ^MIM -binding site is inaccessible in O-Mad2. Time-resolved NMR and molecular dynamics simulations show how Mad2 conversion involves sequential conformational changes of flexible structural elements in O-Mad2, orchestrated by Cdc20 ^MIM . Conversion is initiated by the β7/β8 hairpin of O-Mad2 transiently unfolding to expose a nascent Cdc20 ^MIM -binding site. Engagement of Cdc20 ^MIM to this site promotes release of the β1 strand. We propose that initial conformational changes of the β7/β8 hairpin allows binding of Cdc20 ^MIM to a transient intermediate state of Mad2, thereby lowering the kinetic barrier to Mad2 conversion.

A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein–membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life.

During bacterial cell division, filaments of tubulin-like FtsZ form the Z-ring, which is the cytoplasmic scaffold for divisome assembly. In Escherichia coli, the actin homologue FtsA anchors the Z-ring to the membrane and recruits divisome components, including bitopic FtsN. FtsN regulates the periplasmic peptidoglycan synthase FtsWI. To characterize how FtsA regulates FtsN, we applied electron microscopy to show that E. coli FtsA forms antiparallel double filaments on lipid monolayers when bound to the cytoplasmic tail of FtsN. Using X-ray crystallography, we demonstrate that Vibrio maritimus FtsA crystallizes as an equivalent double filament. We identified an FtsA–FtsN interaction site in the IA–IC interdomain cleft of FtsA using X-ray crystallography and confirmed that FtsA forms double filaments in vivo by site-specific cysteine cross-linking. FtsA–FtsN double filaments reconstituted in or on liposomes prefer negative Gaussian curvature, like those of MreB, the actin-like protein of the elongasome. We propose that curved antiparallel FtsA double filaments together with treadmilling FtsZ filaments organize septal peptidoglycan synthesis in the division plane.

In response to improper kinetochore-microtubule attachments in mitosis, the spindle assembly checkpoint (SAC) assembles the mitotic checkpoint complex (MCC) to inhibit the anaphase- promoting complex/cyclosome, thereby delaying entry into anaphase. The MCC is a complex of Mad2, Cdc20 and BubR1:Bub3. Its assembly is catalysed by unattached kinetochores on a catalytic Mad1:Mad2 platform. Mad1-bound closed-Mad2 (C-Mad2) recruits open-Mad2 (O- Mad2) through self-dimerization. This interaction, combined with Mps1 kinase-mediated phosphorylation of Bub1 and Mad1, accelerates MCC assembly, in a process that requires conversion of O-Mad2 to C-Mad2 and concomitant binding of Cdc20. How Mad1 phosphorylation catalyses MCC assembly is poorly understood. In this study we characterized Mps1 phosphorylation of Mad1 and obtained structural insights into a phosphorylation-specific Mad1:Cdc20 interaction. This interaction, together with the Mps1-phosphorylation dependent association of Bub1 and Mad1, generates a tripartite assembly of Bub1 and Cdc20 onto the C- terminal domain of Mad1 (Mad1 ^CTD ). We additionally identified a folded state of the Mad1:Mad2 complex that suggests how the Cdc20:Mad1 ^CTD interaction brings the Mad2- interacting motif (MIM) of Cdc20 (Cdc20 ^MIM ) into close proximity with O-Mad2. We also show that Cdc20 ^MIM is sufficient to catalyse Mad2 conversion and its entrapment by the Mad2 safety- belt. Thus, Mps1-dependent formation of the MCC-assembly scaffold functions to position and orient Cdc20 ^MIM near O-Mad2, thereby catalysing formation of C-Mad2:Cdc20.

Cell growth and division of walled bacteria depend on the synthesis and remodelling of peptidoglycan (PG). These activities are carried out by two multiprotein complexes, the elongasome and the divisome during cell elongation and division, respectively. Filaments of tubulin-like FtsZ form the cytoplasmic scaffold for divisome assembly, the Z-ring. In E. coli , the actin homologue FtsA anchors the Z-ring to the membrane and recruits downstream divisome components, including bitopic FtsN. FtsN is recruited late and activates the periplasmic PG synthase FtsWI. To start unravelling the activation mechanism involving FtsA and FtsN, we showed that E. coli FtsA forms antiparallel double filaments on lipid monolayers when also binding FtsN’s cytoplasmic tail, and that Vibrio maritimus FtsA crystallised as an equivalent double filament. We structurally located the FtsA-FtsN interaction site in FtsA’s IA-IC interdomain cleft and confirmed FtsA double filament formation in vivo using site-specific cysteine cross-linking. FtsA-FtsN double filaments reconstituted on and in liposomes preferred negative Gaussian curvature, as was previously shown for the elongasome’s actin, MreB. MreB filaments serve as curvature-sensing “rudders”, orienting insertion of PG around the cell’s circumference. We propose that curved antiparallel FtsA double filaments function similarly in the divisome: FtsA filaments, together with dynamic FtsZ filaments orient and concentrate cell-constricting septal PG synthesis in the division plane.

During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1 ^CTD ) bound to two phosphorylated Bub1 ^CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1 ^CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1 ^CTD and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly.