Significance: Lattice light sheet structured illumination microscopy (latticeSIM) has proven highly effective in producing 3D images with super resolution rapidly and with minimal photobleaching. However, due to the use of two separate objectives, sample-induced aberrations can result in an offset between the planes of excitation and detection, causing artifacts in the reconstructed images. Aim: We introduce a posterior approach to detect and correct for the axial offset between the excitation and detection focal planes in latticeSIM and provide a method to minimize artifacts in the reconstruc ..read more

A bilateral body plan is predominant throughout the animal kingdom. Bilaterality of amniote embryos becomes recognizable as midline morphogenesis begins at gastrulation, bisecting an embryonic field into the left and right sides. Soon after, left-right asymmetry also starts. While a series of laterality genes expressed after the left-right compartmentalization has been extensively studied, the laterality patterning prior to and during midline morphogenesis has remained unclear. Here, through a biophysical quantification in a high spatial and temporal resolution, applied to a chick model system ..read more

Electron tomography of frozen, hydrated samples allows structure determination of macro-molecular complexes that are embedded in complex environments. Provided that the target complexes may be localised in noisy, three-dimensional tomographic reconstructions, averaging images of multiple instances of these molecules can lead to structures with sufficient resolution for de novo atomic modelling. Although many research groups have contributed image processing tools for these tasks, a lack of standardisation and inter-operability represents a barrier for newcomers to the field. Here, we present a ..read more

Cyclic nucleotide-binding domain (CNBD) ion channels play crucial roles in cellular-signaling and excitability and are regulated by the direct binding of cyclic adenosine- or guanosine-monophosphate (cAMP, cGMP). However, the precise allosteric mechanism governing channel activation upon ligand binding, particularly the energetic changes within domains, remains poorly understood. The prokaryotic CNBD channel SthK offers a valuable model for investigating this allosteric mechanism. In this study, we investigated the conformational dynamics and energetics of the SthK C-terminal region using a co ..read more

OaPAC is a recently discovered blue-light using flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report the crystal structures of the enzyme in the absence of its natural substrate determined from room temperature serial crystallography data collected at both an X-ray free electron laser and a synchrotron and we compare them with the cryo macromolecular crystallography structures obtained at a synchr ..read more

The vesicle-tethering exocyst complex is a key regulator of cell polarity. The subunit Exo70 is required for the targeting of the exocyst complex to the plasma membrane. While the N-terminus of Exo70 is important for its regulation by GTPases, the C-terminus binds to PI(4,5)P2 and Arp2/3. Here, we compare N- and C-terminal tagged Exo70 with respect to subcellular localization, dynamics and function in cell membrane expansion. Using high-resolution imaging, we determined the spatial distribution and dynamics in different sub-compartments of un-polarized and polarized cells. With lattice light-s ..read more

Eukaryotic translation initiation factor (eIF) 3 is a multi-subunit protein complex that binds both ribosomes and messenger RNAs (mRNAs) in order to drive a diverse set of mechanistic steps during translation. Despite its importance, a unifying framework explaining how eIF3 performs these numerous activities is lacking. Using single-molecule light scattering microscopy, we demonstrate that Saccharomyces cerevisiae eIF3 is an equilibrium mixture of the full complex, subcomplexes, and subunits. By extending our microscopy approach to an in vitro reconstituted eIF3 and complementing it with bioch ..read more

Water-mediated proton transfer reactions are central for catalytic processes in a wide range of biochemical systems, ranging from biological energy conversion to chemical transformations in the metabolism. Yet, the accurate computational treatment of such complex biochemical reactions is highly challenging and requires the application of multiscale methods, in particular hybrid quantum/classical (QM/MM) ap-proaches combined with free energy simulations. Here we combine the unique exploration power of new advanced sampling methods with density functional theory (DFT)-based QM/MM free energy met ..read more

The discovery of microbial-derived DNA-interacting agents, which hold broad therapeutic potential, is inherently challenging due to the limited sensitivity and specificity of conventional methodologies. Our study introduces a pioneering application of single-molecule stretching assay (SMSA) in natural product chemistry to identify DNA-intercalating agents directly from microbial cultures or extracts. We demonstrate that mechanical force can enhance sensitivity by increasing both the binding affinity Ka and the quantity of ligands bound. The changes induced by intercalators in the counter lengt ..read more

The adaptor protein human GRB2 plays crucial roles in mediating signal transduction from cell membrane receptors to RAS and its downstream proteins by recruiting SOS1. Recent studies have revealed that GRB2 also serves as a scaffold for liquid-liquid phase separation (LLPS) with SOS1 and transmembrane receptors, which is thought to regulate the magnitude of cell signalling pathways. In this study, we employed solution NMR spectroscopy to investigate the interactions of the full-length GRB2 with proline-rich motifs (PRMs) derived from ten potential GRB2-binding sites in SOS1, as well as a pepti ..read more