Optimizing cryogenic super-resolution microscopy for integrated structural biology
Super-resolution fluorescence microscopy (“nanoscopy”) enables biological imaging at the nanoscale. This technique has already revolutionized cell biology, and today it enters the field of structural biology. One major evolution concerns the development of nanoscopy at cryogenic temperature (“cryo-nanoscopy”). Cryo-nanoscopy offers several key advantages, notably the prospect of an extremely precise correlation with cryo-electron tomography (cryo-ET) data. However, cryo-nanoscopy has not provided super-resolved images of sufficiently high quality yet. This PhD project will focus on the optimization of cryo-nanoscopy using the Single Molecule Localization Microscopy (SMLM) method with fluorescent proteins (FPs) as markers. Our goal is to significantly improve the quality of achievable cryo-SMLM images by (i) engineering and better understanding the photophysical properties of various FPs at cryogenic temperature, (ii) modifying a cryo-SMLM microscope to collect better data and (iii) developing the nuclear pore complex (NPC) as a metrology tool to quantitatively evaluate cryo-SMLM performance. These developments will foster cryo- correlative (cryo-CLEM) studies linking cryo-nanoscopy and cryo-FIB-SEM-based electron tomography.
INVESTIGATION OF CONFORMATIONAL HETEROGENEITY AND DYNAMICS IN FLUORESCENCE ACTIVATING AND ABSORPTION-SHIFTING TAGS (FAST)
Fluorescent proteins, particularly Reversibly Switchable Fluorescent Proteins (RSFPs), have revolutionized advanced fluorescence imaging, paving the way for applications such as super-resolution microscopy. Among emerging alternatives, fluorogen-based reporters, such as the FAST (Fluorescence Activating and Absorption Shifting Tag) system, stand out dur to their enhanced photostability and versatility. FAST operates via non-covalent binding of a small engineered protein to an organic fluorogen, which induces fluorescence and allowing real-time monitoring without chromophore maturation. However, challenges remain in optimizing these systems due to limited mechanistic understanding of fluorogen-protein interactions, binding dynamics, and photophysical behavior under illumination. This PhD project aims to characterize the binding modes of FAST systems at atomic resolution using multidimensional NMR spectroscopy, X-ray crystallography, and UV-visible spectroscopy. Recent findings suggest that fluorogens can adopt multiple binding modes, and that slight chemical modifications impact binding kinetics and fluorescence brightness. By integrating laser-based illumination in NMR investigations, we will further probe how light absorption affects fluorogen conformation and dynamics. The insights gained from this study will enable the rational design of optimized FAST variants, enhancing their performance for specific microscopy applications and advancing the field of fluorescence imaging.
Molecular dynamics and disorder in the viral replication machinery of SARS CoV 2
The nucleocapsid protein (N) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for genome replication, encapsidating the viral genome and regulating gene transcription. The protein is highly disordered, comprising two disordered termini and a central disordered domain that are essential to its function. The central domain contains a number of important mutations that are responsible for enhanced viral fitness, and comprises a region that is hyperphosphorylated during the viral cycle. NMR spectroscopy is the tool of choice for studying the conformational behaviour of intrinsically disordered proteins, an abundant class of proteins that are functional in their disordered form. They represent 40% of the proteome and are too dynamic to be studied by crystallography or electron microscopy. The host lab has developed a large number of unique NMR-based tools to help understand the function of this class of proteins at atomic resolution. We will use NMR, paramagnetic NMR, small angle scattering, single molecule FRET and electron microscopy, in combination with molecular dynamics simulation, to describe the interactions of N with viral partner proteins and viral RNA to describe the process of encapsidation of the viral genome by the nucleocapsid protein, as well as the impact of mutations present in variants of concern. The results will be correlated with light and electron microscopy, carried out in collaboration.
Unraveling the mechanism of enzymatic carbon fixation
The Synchrotron Group at the Institut de Biologie Structurale in Grenoble is currently developing an innovative method called TR-FOX (Time-Resolved Functional Oscillation Crystallography). This technique aims to elucidate, firstly, the global dynamics of biological macromolecules in action and, secondly, their fine catalytic mechanism. It relies on the use of an injector capable of depositing onto the crystal, during the course of the X-ray diffraction data collection, a nanoliter droplet containing the substrate and cofactor of the studied reaction. This triggers the enzymatic reaction within the crystal. The approach can be combined with UV-Visible absorption spectroscopy to characterize the reaction kinetics more precisely. The goal is to obtain a series of structures during the catalytic cycle in order to make a molecular movie depicting the functioning of the enzyme. This thesis has two objectives: (i) improve and validate the TR-FOX method and, (ii) study the catalytic mechanism of two enzymes involved in carbon fixation either by capture or conversion of CO2.
Towards a better understanding of membrane proteins through AI
Despite the remarkable advances in artificial intelligence (AI), particularly with tools like AlphaFold, the prediction of membrane protein structures remains a major challenge in structural biology. These proteins, which represent 30% of the proteome and 60% of therapeutic targets, are still significantly underrepresented in the Protein Data Bank (PDB), with only 3% of their structures resolved. This rarity is due to the difficulty in maintaining their native state in an amphiphilic environment, which complicates their study, especially with classical structural techniques.
This PhD project aims to overcome these challenges by combining the predictive capabilities of AlphaFold with experimental small-angle scattering (SAXS/SANS) data obtained under physiological conditions. The study will focus on the translocator protein TSPO, a key marker in neuroimaging of several serious pathologies (cancers, neurodegenerative diseases) due to its strong affinity for various pharmacological ligands.
The work will involve predicting the structure of TSPO, both in the presence and absence of ligands, acquiring SAXS/SANS data of the TSPO/amphiphile complex, and refining the models using advanced modeling tools (MolPlay, Chai-1) and molecular dynamics simulations. By deepening the understanding of TSPO’s structure and function, this project could contribute to the design of new ligands for diagnostic and therapeutic purposes.
Dynamic interplay of Rad51 nucleoprotein filament-associated proteins - Involvement in the regulation of homologous recombination
Homologous recombination (HR) is an important repair mechanism for DNA double-strand breaks induced by ionizing radiation. A key step in HR is the formation of Rad51 nucleoprotein filaments on the single-stranded DNA that is generated from these breaks. We were the first to show, using yeast as a model, that a tight control of the formation of these filaments is essential for HR not to induce chromosomal rearrangements by itself (eLife 2018, Cells 2021). In humans, the functional homologs of the yeast control proteins are tumor suppressors. Thus, the control of HR seems to be as important as the mechanism of HR itself. Our project involves the use of new molecular tools that allow a breakthrough in the study of these controls. We will use a functional fluorescent version of the Rad51 protein, first developed by our collaborators A. Taddei (Institut Curie), R. Guérois and F. Ochsenbein (I2BC, Joliot, CEA). This major advance will allow us to observe the influence of regulatory proteins on DNA repair by microscopy in living cells. We have also developed highly accurate structural models of control protein complexes associated with Rad51 filaments. We will adopt a multidisciplinary approach based on genetics, molecular biology, biochemistry, and protein structure in collaboration with W.D. Heyer (University of California, Davis, USA), to understand the function of the regulators of Rad51 filament formation. The description of the organization of these proteins with Rad51 filaments will allow us to develop new therapeutic approaches.
Condensates and Chromatin: How Phase Separation Shapes Plant Temperature Responses
Plants must adapt their development to environmental conditions, including rising temperatures due to climate change. Heat stress significantly impacts plant physiology, and to mitigate these effects, plants have evolved heat shock responses (HSR), with Heat Shock Factor A1a (HSFA1a) serving as a master regulator in Arabidopsis thaliana. Under nonstress conditions, HSFA1a remains cytosolic and inactive, bound to heat shock proteins (HSPs). Heat stress triggers HSP dissociation, enabling HSFA1a nuclear translocation, trimerization, chromatin binding, and activation of stress-responsive genes. Recent studies reveal that HSFA1a might act as a pioneer transcription factor to access closed chromatin regions and initiate HSR. Additionally, preliminary findings also suggest that HSFA1a undergoes liquid-liquid phase separation (LLPS) to form nuclear condensates that regulate gene expression. This project aims to 1) explore how temperature affects HSFA1a structure and oligomerization, 2) investigate LLPS of HSFA1a with and without DNA, 3) characterize HSFA1a pioneer activity, and 4) determine the physiological importance of LLPS in HSR.
Nitrogenase Active Site Assembly: What Distinguishes a Nitrogenase from a Scaffold
The challenges posed by climate change and soil degradation call for urgent solutions to reduce greenhouse gas emissions and reliance on nitrogen fertilizers while ensuring sufficient crop yields to feed a growing global population. A natural solution lies in the use of nitrogenase, a bacterial enzyme capable of converting atmospheric nitrogen into ammonia, which can be directly assimilated by plants. However, the biosynthesis of its metal cofactor, FeMo-co, is a complex process that requires the coordinated action of numerous proteins.
This PhD project aims to streamline this complex process by studying simplified nitrogenase systems found in certain organisms, which use fewer proteins, notably by combining multiple functions into single proteins. By conducting comparative structural and functional studies, we seek to understand how these simplified systems work and how they can be adapted for use in crops like cereals, potentially allowing large-scale cultivation without heavy nitrogen fertilizer use.
This project is a collaboration between leading teams at CEA’s Institute of Structural Biology and CSIC Madrid, specializing in metalloprotein structure-function analysis and the biochemistry and genetics of nitrogenase assembly. The successful candidate will work in a cutting-edge research environment, gaining international experience and valuable skills for a future career in academic research or R&D.