A cryo light microscope with confocal/super-resolution capabilities to target macromolecular complexes for in-cell structural biology

Structural biology is the field of biology which investigates the three-dimensional (3D) structures of macromolecules, in particular of protein complexes, the main molecular machines which perform cellular functions and allow cellular life. Knowing the structure of protein complexes with atomic details allows scientists to understand its function, since the 3D architecture shows the chemical arrangement of aminoacids in space, key to interpret how a protein works. Many diseases are linked to defects in protein architecture, protein complex assembly, protein-protein interactions, pathogen's proteins, making the understanding of protein structure fundamental to find means to fight diseases. Classic structural biology methods study the structure of protein complexes extracted from their cellular context, nevertheless the long-term goal of structural biology is to solve the structure of proteins in their cellular milieu, their most physiological environment. In order to achieve such goal, the protein of interest must be found within the cellular environment and thin sections must be cut in the same area to acquire high-resolution images with an electron microscope at very low temperatures (-196C). From such images a 3D volume can be obtained from where proteins must be extracted with computational methods to solve their 3D structure at near atomic resolution.
One of the ways to find the protein of interest in the cellular context is to use a fluorescent tag attached to the protein using molecular biology methods. The instrument funded by this proposal will allow to spot the protein fluorescence with high spatial precision at -196C. Such precise localisation in the frozen sample is fundamental to target the cut of a thin section (150 billionth of a meter thick) for subsequent structural biology characterisation. The instrument to be funded is a cutting-edge light microscope which allow to image the cell of interest at high speed and without provoking any damage to the biological sample.
Several research projects will be made possible from this machine, in the area of molecular and cellular medicine, infection and antibiotic resistance, and neurobiology, all relevant areas to the MRC.
The microscope will be used to understand the structure of the protein complexes involved in chromosomes segregation in mammalian and yeast cells (Barford lab), fundamental cellular process to understand how a cell allows a correct distribution of chromosomes after each division. The structural knowledge will be therefore critical to find therapeutic targets in case of defects in this protein machinery, since such defects are a hallmark of cancer cells.
The microscope will be also used to investigate the mechanism of bacterial cell division (Löwe lab), a conserved process in all microbial world that can be tackled to fight infectious bacteria when structural information will be available.
Bharat lab will instead use the instrument to investigate the formation and organisation of bacterial biofilms at the molecular level, a fundamental mechanism of infection used by many pathogenic bacteria such as P. aeruginosa, which is a major cause of antibiotic-tolerant infections in humans and has been listed as a critical priority for the research of new treatments by the World Health Organisation.
Allegretti lab will use the instrument to investigate the architecture of nuclear remodelling and repair's machineries, critical protein complexes that allow cell migration, but if dysregulated they favour cancer metastatic progression and, if mutated, they cause premature ageing.
In summary the acquisition of such an instrument will tremendously support the research plans of the Laboratory of Molecular Biology (LMB) with more than 30 users and already 9 projects planned in the first year after machine's acquisition. LMB scientists are confident that this microscope will become therefore a fundamental tool to enhance the structural biology goals of the LMB

The next frontier of structural biology is to obtain atomic resolution structures of protein complexes in their cellular environment. Electron cryo tomography (cryo-ET) allows to obtain high-resolution snapshots of the cellular context and obtain near atomic resolution protein structures. The requested cryo confocal/superresolution LSM 900 helps to localise with high spatial precision (290nm in X-Y direction and 1um in Z direction) the macromolecule of interest thanks to the Airyscan 2 module, which improves confocal resolution by a factor of up to 1.5 under cryogenic conditions while still providing high signal-to-noise image stacks. Those stacks can be used to precisely cut a 200nm lamella with a cryo-Focused Ion Beam (FIB)-milling machine upon image correlation using fluorescent beads and internal fluorescent markers. The software ZEN connect is an integrated software between the cryo-confocal and the cryo-FIB-milling that allows an easy correlation between the two imaging modalities. The transition between the two machines is also facilitated by the usage of the same cryo-holder which highly reduce the risks of contaminating the vitrified sample. Finally the lamellae will be transferred to a 300kV Titan Krios for acquisition of high-resolution tilt-series using a K3 detector and the data will be processed to get high-resolution structures using the Warp/Relion/M software.
Many research projects will be carried out with the machine enhancing the in situ cryo-EM portfolio of the LMB. For instance the investigation of the structure of kinethocores involved in the microtubule-based mechanism of chromosomes segregation; the structure of Type I fimbriae from E. coli and Type IV pili from P. aeruginosa which mediate surface adhesion, first step of biofilm formation; the structure of the conserved Ftsz/FtszA/peptidoglycan assembly, which permits cell division in bacteria; and six other projects which are ready to be performed after the microscope's purchase