NanoImaging Services

Structural Biology has significantly influenced drug discovery and development over the past few decades.The use of structural information to design and improve drug molecules trace back to the early 1980s, following the advent of macromolecular X-ray crystallography.In the 1990s significant advancements in computational power and algorithms greatly enhanced the capability of Structure Based Drug Design (SBDD).In the following years, integration of structural information with high-throughput screening, computational chemistry, and chemical biology underscored the potential of SBDD to produce highly effective and specific drugs.Most recently, the incorporation of cryo-electron microscopy (cryo-EM) as structural tool has enabled high-resolution structure determination of systems previously inaccessible thus expanding the space of accessible targets and the breadth of structure-based drug designThe transformation of SBDD from a niche approach to a mainstream strategy in drug discovery can be clearly seen through the several SBDD courses held in Erice (TP) since 1983 as part of the International School of Crystallography.This presentation will provide an overview of the advancements in SBDD (from small molecule design to integration with AI) and will cover pivotal moments in its history as conveyed through these educational sessions, showcasing its transformation and its significant impact on modern therapeutic development.

Abstract Antimicrobial resistance represents a major threat to human health andPseudomonas aeruginosastands out among the pathogens responsible for this emergency. The SOS response to DNA damage plays a pivotal role in bacterial evolution, driving the development of resistance mechanisms and influencing the adaptability of bacterial populations to challenging environments, particularly in the context of antibiotic exposure. Recombinase A (RecA) and the transcriptional repressor LexA are the key players that orchestrate this process, determining either the silencing or the active transcription of the genes under their control. By integrating state-of-the-art structural approaches with binding and functional assaysin vitro, we elucidated the molecular events governing the SOS response activation inP. aeruginosa, focusing on the RecA-LexA interaction. Our findings identify the conserved determinants and strength of the interactions that let RecA trigger the autocleavage and inactivation of the LexA repressor. These results provide the groundwork for designing novel antimicrobial strategies and for exploring the potential translation ofEscherichia coli-derived approaches, to address the health-threatening implications of bacterial infections.Graphical Abstract

As a service provider of small molecule structure determination by MicroED, we have seen the gamut of highquality structures with coordinates indistinguishable from scXRD to highly problematic structures with substantial disorder and/or those requiring significant restraints. Even the question of how to tell a problematic structure from a highly-quality structure is complicated by the generally higher R-factors produced by MicroED structures refined kinematically compared to those from scXRD. Not all MicroED structures with high R-factors are bad and relatively low R-factors don't always indicate a high-quality structure. In this short talk, I will present some of our recent structures and discuss how to think about the quality of MicroED structures and what conclusions can be drawn from them.

Key proteins of retroviruses and other RNA viruses are translated and subsequently processed from polyprotein precursors by the viral protease (PR). Processing of the HIV Gag-Pol polyprotein yields the HIV structural proteins and enzymes. Structures of the mature enzymes PR, reverse transcriptase (RT), and integrase (IN) aided understanding of catalysis and design of antiretrovirals, but knowledge of the Pol precursor architecture and function before PR cleavage is limited. We developed a system to produce stable HIV-1 Pol and determined its cryo–electron microscopy structure. RT in Pol has a similar arrangement to the mature RT heterodimer, and its dimerization may draw together two PR monomers to activate proteolytic processing. HIV-1 thus may leverage the dimerization interfaces in Pol to regulate assembly and maturation of polyprotein precursors.

Transmission electron microscopy has been an important research tool for many decades: the first transmission electron microscope was developed in 1931 by Max Knoll and Ernst Ruska, and in 1981 Jacques Dubochet and Alasdair McDowall introduced the concept of cryo-cooling individual molecules allowing them to retain their native shape and protecting them from environmental and radiation damage 1 . 4] But while cryoEM has proven to be effective in the early stages of drug development, and its applicability to small molecule lead identification and optimization has also been recently demonstrated

Human Arginase 1 (hArg1) is a metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea and modulates T-cell-mediated immune response. Arginase-targeted therapies have been pursued across several disease areas including immunology, oncology, nervous system dysfunction, and cardiovascular dysfunction and diseases. Currently, all published hArg1 inhibitors are small molecules usually less than 350 Da in size. Utilizing cryo-electron microscopy we have obtained structures of potent and inhibitory anti-hArg antibodies bound to hArg1. These distinct macromolecular complexes are made up of multiple hArg and mAb molecules and are greater than 650 kDa in size. With local resolutions of 3.5 or better we unambiguously mapped epitopes and paratopes for all five antibodies and determined that the antibodies act through both orthosteric and allosteric mechanisms. These hArg1:antibody complexes for the first time present an alternative mechanism to inhibit hArg1 activity and highlight the ability to utilize antibodies as probes in the discovery and development of peptide and small molecule inhibitors for enzymes in general.

Igs in vertebrates comprise equally sized H and L chains, with exceptions such as H chain-only Abs in camels or natural Ag receptors in sharks. In Reptilia, Igs are known as IgYs. Using immunoassays with isotype-specific mAbs, in this study we show that green turtles (Chelonia mydas) have a 5.7S 120-kDa IgY comprising two equally sized H/L chains with truncated Fc and a 7S 200-kDa IgY comprised of two differently sized H chains bound to L chains and apparently often noncovalently associated with an antigenically related 90-kDa moiety. Both the 200- and 90-kDa 7S molecules are made in response to specific Ag, although the 90-kDa molecule appears more prominent after chronic Ag stimulation. Despite no molecular evidence of a hinge, electron microscopy reveals marked flexibility of Fab arms of 7S and 5.7S IgY. Both IgY can be captured with protein G or melon gel, but less so with protein A. Thus, turtle IgY share some characteristics with mammalian IgG. However, the asymmetrical structure of some turtle Ig and the discovery of an Ig class indicative of chronic antigenic stimulation represent striking advances in our understanding of immunology.