Tubulin nucleation and microtubule (MT) assembly are frequent events in cells. The mechanisms directing these processes are poorly understood, owing to the size of tubulin and its highly dynamic character. Tau proteins that stabilize MT, are major determinants of cytoskeleton stability. Malfunctions of tubulin and tau are involved in various pathologies including cancer and neurodegenerative diseases. We propose to reconstitute in vitro minimal model systems that mimic these key elements of the cytoskeleton, and follow, in real-time, their coassembled dynamic structures, stability, and interactions. Using SAXS and time-resolved SAXS we will determine the structures and intermolecular forces dictating the various ways tubulin dimers dynamically nucleate, assemble, and interact with one another to stabilize and form MT.

The Israeli BAG plans to increase its usage of ESRF in the coming year. We are having more projects that utilize the ESRF single crystal capabilities as well as the continuous increase in SAXS time. As mentioned last year It is not trivial to provide a summary that covers all research topics encompassed by the BAG. We expect some BAG members to resume using ESRF after utilizing other facilities. As much as we appreciate the immense capacities of ESRF, the majority of BAG members are discontent (to say the least) regarding the post-shutdown/Covid-19 travel regulations imposed by the management. It seems that bureaucracy rather than common sense is prevailing and an accompanying letter will be submitted adjoined to the proposal/report. I have not included any requests for ID29 as the BAG members have to become more familiarized with the facility. As for some of the BAG projects, a detailed description is provided in the Global Summary below.

We propose to unravel the precise assembly and disassembly mechanism of Cp183 and the coassembly mechanism of RNA with Cp183 capsid protein into mature paricles under different solution conditions. The detailed structural changes will be studied at msec temporal resolution. We will characterize the structure, kinetics, mechanism, and energetics of the assembly and disassembly processes under different conditions. To distinguish between different theoretical kinetic models, we need to vary the initial conditions of the assembly and disassembly reactions. Varying the salt concentration or pH has a critical role on the assembly and disassembly processes, as these factors determine the strength of the interaction between protein capsid molecules and between the RNA and the capsid protein.

The Israeli BAG plans to increase its usage of ESRF in the coming year. We are having more projects that utilize the ESRF single crystal capabilities as well as the continuous increase in SAXS time. As mentioned last year It is not trivial to provide a summary that covers all research topics encompassed by the BAG. We expect some BAG members to resume using ESRF after utilizing other facilities. As much as we appreciate the immense capacities of ESRF, the majority of BAG members are discontent (to say the least) regarding the post-shutdown/Covid-19 travel regulations imposed by the management. It seems that bureaucracy rather than common sense is prevailing and an accompanying letter will be submitted adjoined to the proposal/report. I have not included any requests for ID29 as the BAG members have to become more familiarized with the facility. As for some of the BAG projects, a detailed description is provided in the Global Summary below.

Microtubules (MTs) are one of the major components of the cytoskeleton, involved in cell division, intracellular transport, cell motility, and cell shape. Precise control of the highly-frequent events of tubulin nucleation is needed for a proper cytoskeleton function. Despite years of research, tubulin nucleation is still one of the least understood steps of MT dynamics. To determine the molecular mechanisms directing tubulin nucleation, we will reconstitute in-vitro a minimal model system that mimics the key elements of non-templated cytoskeleton MT assembly. Using state-of-the-art time-resolved SAXS (TR-SAXS) and our home-developed cutting-edge analysis tools, we shall follow, in real-time, the active self-organization, dynamic structures and energetics of tubulin protein subunits and their regulation by nucleotide hydrolysis. These data will resolve the underlying mechanisms at the molecular level by which nucleotides, tubulin, and MT assemble and function.

The Israeli BAG plans to increase its usage of ESRF in the coming year. We are having more projects that utilize the ESRF single crystal capabilities as well as the continuous increase in SAXS time. As mentioned last year It is not trivial to provide a summary that covers all research topics encompassed by the BAG. We expect some BAG members to resume using ESRF after utilizing other facilities. As much as we appreciate the immense capacities of ESRF, the majority of BAG members are discontent (to say the least) regarding the post-shutdown/Covid-19 travel regulations imposed by the management. It seems that bureaucracy rather than common sense is prevailing and an accompanying letter will be submitted adjoined to the proposal/report. I have not included any requests for ID29 as the BAG members have to become more familiarized with the facility. As for some of the BAG projects, a detailed description is provided in the Global Summary below.

The BAG is quite large with 24 principal investigators covering almost all groups in Israel. The scientific topics are diverse and so different that depicting them in a short Abstract is extremely difficult. In this context BAG members are studying the parameters that govern protein assemblage, structural impacts of signaling processes in different cascades. There are several projects that are directed towards molecular recognition with implementation towards drug design. In addition, we still have the ribosome project and the design of unique antibiotics with specific functional interactions from the mycinamycins family and their resistance related mechanism compared with other macrolides and the photosystem and how it converts light into energy. Since Covid-19 is still a relevant topic as are other viral infections BAG members are and have been involved in the understanding of the infection process and the means to hinder it.

This Summary will only portray a very limited scope of the BAG proposal due to its size and more information will be available in the Global Summary. We plan establish a comprehensive structural narrative for the assembly and entry of viruses from the Bunyavirales family using X-ray crystallography, biophysics, biochemistry, and molecular virology. Using X-ray crystallography to obtain the 3D structure of GN and GC in different conformations and from different bunyavituses. We will pursue a structure of GN or GC bound to a neutralizing antibody to obtain new data for vaccine design. In the SAXS arena, we plan to continue the study on MT (microtubules) and expand the experiments with different nucleotide type and concentrations, (GTP, GDP, GMPCPP, and GMPPCP, GTP-gammaS). We will perform at selected points SEC-SAXS experiments in the presence of full-length tau (4RL). Our preliminary data revealed that 4RL promote MT assembly at 36C and of single tubulin rings at low temperatures.

Tubulin nucleation and microtubule (MT) assembly are frequent events in cells. The mechanisms directing these processes are poorly understood, owing to the size of tubulin and its highly dynamic character. The MT-associated tau proteins, including six isoforms, are major determinants of axon cytoskeleton stability and dynamics. Malfunctions of tubulin and tau are involved in various pathologies including cancer and neurodegenerative diseases. We propose to use time-resolved solution small-angle-X-ray scattering (TR-SAXS) methods, and our advanced analysis tools to follow the coassembly dynamics of different tubulin with different tau isoforms. Our aim is to determine the structures and intermolecular interactions dictating how tubulin dimers and different tau isoforms dynamically nucleate, assemble, and interact with one another to form tau-stabilized MT.