Annual Review 2024-2025

D I A M O N D L I G H T S O U R C E L I M I T E D B I O L O G I C A L C RY O - I M A G I N G G R O U P 09 The Biological Cryo-Imaging Group brings together dedicated facilities for X-ray, light, and electron microscopy at Diamond. The electron Bio-Imaging Centre (eBIC) is a CryoEM centre providing scientists with state-of- the-art experimental equipment and expertise in the field of cryo-electron microscopy, for single particle analysis, electron tomography and electron diffraction. The location of eBIC enables scientists to combine their techniques with many of the other cutting-edge approaches that Diamond offers. Currently eBIC houses five Titan Krios microscopes, a Talos Arctica, two Glacios microscopes, Scios and Aquilos cryo-FIB/SEMs, and a Leica CryoCLEM. Beamline B24 hosts a full field cryo-transmission X-ray microscope dedicated to biological X-ray imaging and has also established a cryo super resolution fluorescence microscopy facility, which is a joint venture between Diamond and the University of Oxford. It provides a unique platform for correlative light and X-ray microscopy, and cryoEM. A recent external beamline review rated the B24 facility as excellent and world leading. In particular, the panel commended the beamline team on establishing an internationally unique correlative platform combining two high-end 3D cryomicroscopy techniques (Cryo Soft X-ray Tomography (CryoSXT) and Cryo Structured Illumination Microscopy (CryoSIM) with user friendly protocols. Jigsaw puzzle: Deciphering the chloroplast transcription machinery Chloroplasts are specialised organelles found in plant cells and some algae. Chloroplasts have a unique transcription machinery that is more complex than their cyanobacterial ancestors. The plastid-encoded RNA polymerase (PEP) is a multi-subunit complex crucial for transcribing chloroplast genes, which are essential for photosynthesis and plant growth. Despite its importance, the roles of many PEP- associated proteins (PAPs) are poorly understood. In this study, researchers from the John Innes Centre purified PEP complex using chromatographic separation. It showed that the PEP is a huge complex of 1.1 MDa, more than twice the size of its bacterial counterpart. To visualise the PEP complex at high resolution, the researchers used cryoEM at eBIC. CryoEM is ideal for studying large protein complexes in their native state, allowing the researchers to capture the intricate details of PEP and its associated proteins. The PEP complex in chloroplasts. Biological Cryo-Imaging Group Plant Chloroplast PEP Complex Superoxide Dismutases RNA Polymerase Amino acid Ligase Thioredoxins RNA-binding DNA-binding Methyltransferase They discovered that the core polymerase of the PEP shares structural similarities with the cyanobacterial RNAP. Also, PAPs encase the core polymerase, forming extensive interactions that likely promote complex assembly and stability. The PAP subunits add new capability to the core polymerase. PAP1 and PAP2 add DNA binding and RNA binding, and several PAPs add enzymatic functions. Interestingly, if any single PAP subunit is missing, the polymerase will not function efficiently.