Structure of a PSI-LHCI-cyt b6f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions.

Photosynthetic linear electron flow (LEF) produces ATP and NADPH, while cyclic electron flow (CEF) exclusively drives photophosphorylation to supply extra ATP. The fine-tuning of linear and cyclic electron transport levels allows photosynthetic organisms to balance light energy absorption with cellular energy requirements under constantly changing light conditions. As LEF and CEF share many electron transfer components, a key question is how the same individual structural units contribute to these two different functional modes. Here, we report the structural identification of a photosystem I (PSI)-light harvesting complex I (LHCI)-cytochrome (cyt) b6f supercomplex isolated from the unicellular alga Chlamydomonas reinhardtii under anaerobic conditions, which induces CEF. This provides strong evidence for the model that enhanced CEF is induced by the formation of CEF supercomplexes, when stromal electron carriers are reduced, to generate additional ATP. The additional identification of PSI-LHCI-LHCII complexes is consistent with recent findings that both CEF enhancement and state transitions are triggered by similar conditions, but can occur independently from each other. Single molecule fluorescence correlation spectroscopy indicates a physical association between cyt b6f and fluorescent chlorophyll containing PSI-LHCI supercomplexes. Single particle analysis identified top-view projections of the corresponding PSI-LHCI-cyt b6f supercomplex. Based on molecular modeling and mass spectrometry analyses, we propose a model in which dissociation of LHCA2 and LHCA9 from PSI supports the formation of this CEF supercomplex. This is supported by the finding that a Δlhca2 knockout mutant has constitutively enhanced CEF.

RAZA: A Rapid 3D z-crossings algorithm to segment electron tomograms and extract organelles and macromolecules.

Resolving the 3D architecture of cells to atomic resolution is one of the most ambitious challenges of cellular and structural biology. Central to this process is the ability to automate tomogram segmentation to identify sub-cellular components, facilitate molecular docking and annotate detected objects with associated metadata. Here we demonstrate that RAZA (Rapid 3D z-crossings algorithm) provides a robust, accurate, intuitive, fast, and generally applicable segmentation algorithm capable of detecting organelles, membranes, macromolecular assemblies and extrinsic membrane protein domains. RAZA defines each continuous contour within a tomogram as a discrete object and extracts a set of 3D structural fingerprints (major, middle and minor axes, surface area and volume), enabling selective, semi-automated segmentation and object extraction. RAZA takes advantage of the fact that the underlying algorithm is a true 3D edge detector, allowing the axes of a detected object to be defined, independent of its random orientation within a cellular tomogram. The selectivity of object segmentation and extraction can be controlled by specifying a user-defined detection tolerance threshold for each fingerprint parameter, within which segmented objects must fall and/or by altering the number of search parameters, to define morphologically similar structures. We demonstrate the capability of RAZA to selectively extract subgroups of organelles (mitochondria) and macromolecular assemblies (ribosomes) from cellular tomograms. Furthermore, the ability of RAZA to define objects and their contours, provides a basis for molecular docking and rapid tomogram annotation.

Environmental pathways affecting gene expression (E.PAGE) as an R package to predict gene-environment associations.

The purpose of this study is to manually and semi-automatically curate a database and develop an R package that will act as a comprehensive resource to understand how biological processes are dysregulated due to interactions with environmental factors. The initial database search run on the Gene Expression Omnibus and the Molecular Signature Database retrieved a total of 90,018 articles. After title and abstract screening against pre-set criteria, a total of 237 datasets were selected and 522 gene modules were manually annotated. We then curated a database containing four environmental factors, cigarette smoking, diet, infections and toxic chemicals, along with a total of 25,789 genes that had an association with one or more of gene modules. The database and statistical analysis package was then tested with the differentially expressed genes obtained from the published literature related to type 1 diabetes, rheumatoid arthritis, small cell lung cancer, COVID-19, cobalt exposure and smoking. On testing, we uncovered statistically enriched biological processes, which revealed pathways associated with environmental factors and the genes. The curated database and enrichment tool are available as R packages at https://github.com/AhmedMehdiLab/E.PATH and https://github.com/AhmedMehdiLab/E.PAGE respectively.

A 3D image filter for parameter-free segmentation of macromolecular structures from electron tomograms.

3D image reconstruction of large cellular volumes by electron tomography (ET) at high (≤ 5 nm) resolution can now routinely resolve organellar and compartmental membrane structures, protein coats, cytoskeletal filaments, and macromolecules. However, current image analysis methods for identifying in situ macromolecular structures within the crowded 3D ultrastructural landscape of a cell remain labor-intensive, time-consuming, and prone to user-bias and/or error. This paper demonstrates the development and application of a parameter-free, 3D implementation of the bilateral edge-detection (BLE) algorithm for the rapid and accurate segmentation of cellular tomograms. The performance of the 3D BLE filter has been tested on a range of synthetic and real biological data sets and validated against current leading filters-the pseudo 3D recursive and Canny filters. The performance of the 3D BLE filter was found to be comparable to or better than that of both the 3D recursive and Canny filters while offering the significant advantage that it requires no parameter input or optimisation. Edge widths as little as 2 pixels are reproducibly detected with signal intensity and grey scale values as low as 0.72% above the mean of the background noise. The 3D BLE thus provides an efficient method for the automated segmentation of complex cellular structures across multiple scales for further downstream processing, such as cellular annotation and sub-tomogram averaging, and provides a valuable tool for the accurate and high-throughput identification and annotation of 3D structural complexity at the subcellular level, as well as for mapping the spatial and temporal rearrangement of macromolecular assemblies in situ within cellular tomograms.