@article {159720, title = {Distinct filament morphology and membrane tethering features of the dual FtsZ paralogs in Odinarchaeota.}, journal = {EMBO J}, year = {2025}, month = {2025 Aug 08}, abstract = {

The Asgard phylum has emerged as a model to study eukaryogenesis because of their close relatedness with the eukaryotes. In this study, we use FtsZ proteins from a member of the class Odinarchaeia as representatives to investigate the probable origin, evolution, and assembly of the FtsZ/tubulin protein superfamily in Asgard archaea. We performed a comparative analysis of the biochemical properties and cytoskeletal assembly of FtsZ1 and FtsZ2, the two FtsZ isoforms in the Odinarchaeota metagenome. Our electron microscopy analysis reveals that OdinFtsZ1 assembles into curved single protofilaments, while OdinFtsZ2 forms stacked spiral ring-like structures. Upon sequence analysis, we identified an N-terminal amphipathic helix in OdinFtsZ1, which mediates direct membrane tethering. In contrast, OdinFtsZ2 is recruited to the membrane by the anchor OdinSepF via OdinFtsZ2{\textquoteright}s C-terminal tail. Overall, we report the presence of two distant evolutionary paralogs of FtsZ in Odinarchaeota, with distinct filament assemblies and differing modes of membrane targeting. Our findings highlight the diversity of FtsZ proteins in the archaeal phylum Asgardarchaeota, providing valuable insights into the evolution and differentiation of tubulin-family proteins.

}, issn = {1460-2075}, doi = {10.1038/s44318-025-00529-7}, author = {Kumari, Jayanti and Uthaman, Akhilesh and Bose, Sucharita and Kundu, Ananya and Sharma, Vaibhav and Dutta, Soumyajit and Dhar, Anubhav and Roy, Srijita and Srinivasan, Ramanujam and Pande, Samay and Vinothkumar, Kutti R and Gayathri, Pananghat and Palani, Saravanan} } @article {140645, title = {Exploring advances in single particle CryoEM with apoferritin: From blobs to true atomic resolution.}, journal = {Int J Biochem Cell Biol}, volume = {169}, year = {2024}, month = {2024 Feb 01}, pages = {106536}, abstract = {

Deciphering the three-dimensional structures of macromolecules is of paramount importance for gaining insights into their functions and roles in human health and disease. Single particle cryoEM has emerged as a powerful technique that enables direct visualization of macromolecules and their complexes, and through subsequent averaging, achieve near atomic-level resolution. A major breakthrough was recently achieved with the determination of the apoferritin structure at true atomic resolution. In this review, we discuss the latest technological innovations across the entire single-particle workflow, which have been instrumental in driving the resolution revolution and in transforming cryoEM as a mainstream technique in structural biology. We illustrate these advancements using apoferritin as an example that has served as an excellent benchmark sample for assessing emerging technologies. We further explore whether the existing technology can routinely generate atomic structures of dynamic macromolecules that more accurately represent real-world samples, the limitations in the workflow, and the current approaches employed to overcome them.

}, issn = {1878-5875}, doi = {10.1016/j.biocel.2024.106536}, author = {Premageetha, Gowtham ThambraRajan and Vinothkumar, Kutti R and Bose, Sucharita} } @article {147287, title = {Modeling Ligands into Maps Derived from Electron Cryomicroscopy.}, journal = {J Vis Exp}, year = {2024}, month = {2024 Jul 19}, abstract = {

Deciphering the protein-ligand interactions in a macromolecular complex is crucial for understanding the molecular mechanism, underlying biological processes, and drug development. In recent years, cryogenic sample electron microscopy (cryoEM) has emerged as a powerful technique to determine the structures of macromolecules and to investigate the mode of ligand binding at near-atomic resolution. Identifying and modeling non-protein molecules in cryoEM maps is often challenging due to anisotropic resolution across the molecule of interest and inherent noise in the data. In this article, the readers are introduced to various software and methods currently used for ligand identification, model building, and refinement of atomic coordinates using selected macromolecules. One of the simplest ways to identify the presence of a ligand, as illustrated with the enolase enzyme, is to subtract the two maps obtained with and without the ligand. The extra density of the ligand is likely to stand out in the difference map even at a higher threshold. There are instances, as shown in the case of metabotropic Glutamate receptor mGlu5, when such simple difference maps cannot be generated. The recently introduced method of deriving the Fo-Fc omit map can serve as a tool for validating and demonstrating the presence of the ligand. Finally, using the well-studied β-galactosidase as an example, the effect of resolution on modeling the ligands and solvent molecules in cryoEM maps is analyzed, and an outlook on how cryoEM can be used in drug discovery is presented.

}, keywords = {Cryoelectron Microscopy, Ligands, Models, Molecular}, issn = {1940-087X}, doi = {10.3791/66310}, author = {Jha, Shaileshanand and Bose, Sucharita and Vinothkumar, Kutti R} } @article {135860, title = {Structural snapshots of Mycobacterium tuberculosis enolase reveal dual mode of 2PG binding and its implication in enzyme catalysis.}, journal = {IUCrJ}, volume = {10}, year = {2023}, month = {2023 Nov 01}, pages = {738-753}, abstract = {

Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The underlying mechanism of the 2PG to PEP conversion has been studied in great detail in previous work, however that of the reverse reaction remains to be explored. Here we present structural snapshots of Mycobacterium tuberculosis (Mtb) enolase in apo, PEP-bound and two 2PG-bound forms as it catalyzes the conversion of PEP to 2PG. The two 2PG-bound complex structures differed in the conformation of the bound product (2PG) viz the widely reported canonical conformation and a novel binding pose, which we refer to here as the alternate conformation. Notably, we observed two major differences compared with the forward reaction: the presence of Mg is non-obligatory for the reaction and 2PG assumes an alternate conformation that is likely to facilitate its dissociation from the active site. Molecular dynamics studies and binding free energy calculations further substantiate that the alternate conformation of 2PG causes distortions in both metal ion coordination and hydrogen-bonding interactions, resulting in an increased flexibility of the active-site loops and aiding product release. Taken together, this study presents a probable mechanism involved in PEP to 2PG catalysis that is likely to be mediated by the conformational change of 2PG at the active site.

}, keywords = {Catalysis, Models, Molecular, Mycobacterium tuberculosis, Phosphopyruvate Hydratase, Protein Conformation}, issn = {2052-2525}, doi = {10.1107/S2052252523008485}, author = {Ahmad, Mohammed and Jha, Bhavya and Bose, Sucharita and Tiwari, Satish and Dwivedy, Abhisek and Kar, Deepshikha and Pal, Ravikant and Mariadasse, Richard and Parish, Tanya and Jeyakanthan, Jeyaraman and Vinothkumar, Kutti R and Biswal, Bichitra Kumar} } @article {81591, title = {Quaternary variations in the structural assembly of N-acetylglucosamine-6-phosphate deacetylase from Pasteurella multocida.}, journal = {Proteins}, year = {2020}, month = {2020 Aug 31}, abstract = {

N-acetylglucosamine 6-phosphate deacetylase (NagA) catalyzes the conversion of N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate in amino sugar catabolism. This conversion is an essential step in the catabolism of sialic acid in several pathogenic bacteria, including Pasteurella multocida, and thus NagA is identified as a potential drug target. Here, we report the unique structural features of NagA from P. multocida (PmNagA) resolved to 1.95 {\r a}. PmNagA displays an altered quaternary architecture with unique interface interactions compared to its close homolog, the Escherichia coli NagA (EcNagA). We confirmed that the altered quaternary structure is not a crystallographic artefact using single particle electron cryo-microscopy. Analysis of the determined crystal structure reveals a set of hot-spot residues involved in novel interactions at the dimer-dimer interface. PmNagA binds to one Zn ion in the active site and demonstrates kinetic parameters comparable to other bacterial homologs. Kinetic studies reveal that at high substrate concentrations (~10-fold the K ), the tetrameric PmNagA displays hysteresis similar to its distant neighbor, the dimeric Staphylococcus aureus NagA (SaNagA). Our findings provide key information on structural and functional properties of NagA in P. multocida that could be utilized to design novel antibacterials. This article is protected by copyright. All rights reserved.

}, issn = {1097-0134}, doi = {10.1002/prot.25996}, author = {Manjunath, Lavanyaa and Coombes, David and Davies, James and Dhurandhar, Mugdha and Tiwari, Vikas R and Dobson, Renwick C J and Sowdhamini, R and Ramaswamy, S and Bose, Sucharita} }