Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

The ability to determine time resolved structures of macromolecules carrying out their functions is opening a new frontier in life sciences. A key technique is time resolved serial X-ray crystallography whereby many tiny crystals are sequentially delivered into the X-ray beam at a defined time point after an enzyme reaction or other biological process is initiated. By varying the time delay between initiation and data collection, a high-resolution time-dependent molecular movie of the protein is produced. X-ray Free Electron Lasers (XFELs) produce extremely short (fs) X-ray pulses enabling time delays as short as femtoseconds. Such experiments are highly challenging due to difficulty in obtaining beamtime (and infrequent access) at expensive distant facilities, large teams required, unfamiliar sample preparation and challenging data analysis. Serial synchrotron crystallography (SSX) is democratising time-resolved structural biology by addressing these challenges. SSX can be offered regularly to many more research teams at more local facilities, greatly lowering the entry barrier and can in principle address most timescales of relevance to biology, except the very fastest reactions which inherently require XFEL sources. Many biological processes such as enzyme catalysis occur in the range 10ms-10 microseconds: a time domain currently inaccessible to SSX at Diamond. Increased fluxes at synchrotrons allow diffraction to be obtained in ever thinner time-slices but this cannot be accurately recorded using the current generation of photon counting detectors. This proposal will fund an integrating detector for time resolved macromolecular crystallography (MX) at Diamond allowing the UK community to directly access tens of microsecond-plus timescales using SSX. This would significantly increase UK capabilities/reach of the UK dynamic structural biology community. Key advantages include carrying out many currently XFEL-only experiments using more accessible synchrotrons, with regular access allowing for iterative experiments using a well-defined X-ray source. We seek support to purchase a Jungfrau 9M integrating detector enabling full exploitation of the capability of Diamond to access shorter time regimes. The detector will be installed alongside an existing counting detector at the recently upgraded beamline I24 to take advantage of world leading sample delivery systems and data processing expertise. Our aims include: Integrate the detector into a world leading synchrotron beamline Use samples from the coinvestigator team to provide new insights into enzyme mechanism in a variety of systems by obtaining faster reaction time points Following commissioning offer the use of the Jungfrau detector broadly to the UK and international structural biology academic and industrial communities through Diamond's peer-reviewed and proprietary access mechanisms The Jungfrau 9M will enable us to transcend the current limitations of experiments on multiple biological systems studied by the coinvestigators together with the broader UK and international communities. Projects from the applicant team that would immediately benefit include: Proteins important in antimicrobial resistance Iron containing proteins relevant to industrial biotechnology Understanding how exposure of a protein in the human eye to UV light leads to cataract formation Enzymes involved in the global nitrogen cycle (and so highly relevant to agriculture) where crucial intermediate structures are currently inaccessible without XFEL access.

Chemotaxis is a common biological behavior that allows living organisms to move towards chemical stimuli and it has served as a model for the study of cellular sensory signal transduction and motile behavior. Escherichia coli's basic chemotaxis machinery is the most well-characterized signal transduction system, and it serves as a great tool for studying molecular processes involved in chemotaxis. E. coli cells' sensory machinery is a highly organized array of hundreds of basic core signaling units comprised of three essential components: transmembrane chemoreceptors, histidine kinase, and adaptor protein. Such organization allows cells to amplify and integrate many different signals to find optimal growth circumstances. It is necessary to characterize the precise interactions between the core signaling components in the array and in the native cellular context to understand the molecular processes of chemosensory array formation, activation, and high cooperativity. The most critical part of this project is to reveal the sequential conformational changes during signaling. To do so, the time-resolved method will be developed and combined with cryoelectron tomography for in situ structural analysis. The project aims for a molecular movie of chemotaxis signaling events at nearatomic resolution.

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.