Biology subjects

Agam, G.

Publications and source records attributed to Agam, G..

5 recordsLinked to original sources

bioRxiv2026

Symmetry-Based Center and Rotation Refinement for Fiber Diffraction Patterns

X-ray fiber diffraction patterns exhibit four-fold symmetry that can be exploited, through folding and averaging, to improve signal-to-noise ratio. Accurate folding requires a precise sub-pixel estimate of the symmetry center and precise orientation of the meridional pattern axis to the fiber axis: small center or angular errors blur diffraction features, reduce layer-line sharpness, and introduce errors in spacing measurements. A pixel-level estimate is often too imprecise for this purpose, and detector gaps further complicate the alignment objective. We formulate the masked quadrant-folding problem, define a four-quadrant symmetry loss that consistently excludes invalid pixels, and evaluate several refinement strategies: hierarchical coarse-to-fine grid search; ECC-based rigid registration with global center/orientation correction fitting; ECC registration followed by local gradient refinement; and a hybrid that appends a local grid search on a cropped pattern. Direct gradient optimization from the rough QF alignment was found to be unreliable. Grid search provides a robust, interpretable baseline that directly minimizes the folding objective but is substantially slower than registration; ECC gives a fast near-correct alignment, and the hybrid closes the accuracy gap to brute-force search at a fraction of its runtime. On real datasets with calibration data, applying a calibration center with optimized rotation is effectively optimal. The hybrid center-refinement method has been integrated into the MuscleX package.

biophysics↗

bioRxiv2026

Continuous target-specific mutagenesis and rapid gene evolution by diversity-generating retroelements in Escherichia coli

Sequence-programmable directed evolution systems have great potential to accelerate bioengineering. Diversity-generating retroelements (DGRs) are natural hypermutation systems widely distributed in prokaryotes and bacteriophages with the capacity to introduce diverse mutations at template-specified sites of target genes. Here, we show that DGRs can be installed in E. coli and reprogrammed for the continuous, iterative mutagenesis of user-defined target genes. We show that the DGR template RNA can be reprogrammed for gene- and residue- specific mutagenesis, leaving untargeted, adjacent residues unchanged. Furthermore, we establish continuous DGR-enabled mutagenesis with F-plasmid-mediated horizontal gene transfer of target genes (HGT-DGR) into a new host for the progressive accumulation of target- specific mutations. Iterative HGT-DGR mutagenesis over seven cycles yielded an average mutation load of approximately 6% across adenine positions in the target segment, generating a diverse library of variants comprising 40% mutant sequences, with a median pairwise Hamming distance of 4 among mutant variants. HGT-DGR enables iterative diversification of either the same or different user-specified segments of the target gene, as demonstrated with the directed evolution of the M. mazei pyrrolysyl-tRNA synthetase for non-canonical amino acid incorporation. HGT-DGR provides a simple, low-cost, sequence-programmable system that enables iterative, position-specific and tunable in vivo mutagenesis of any target sequence for applications in biotechnology and medicine.

synthetic biology↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2021

Nanoscale organization of the endogenous ASC speck

The NLRP3 inflammasome is a central component of the innate immune system. Its activation leads to the formation of a supramolecular assembly of the inflammasome adaptor protein ASC, commonly referred to as the "ASC speck". Different models of the overall structure of the ASC speck, as well as the entire NLRP3 inflammasome, have been reported in the literature. While many experiments involve overexpression or in vitro reconstitution of recombinant ASC, the cytoplasmic endogenous ASC speck remains difficult to study due to its relatively small size and structural variability. Here, we use a combination of fluorescence imaging techniques including dual-color 3D super-resolution imaging (dSTORM and DNA-PAINT) to visualize the endogenous ASC speck following NLRP3 inflammasome activation. We observe that the complex varies in diameter between [~]800 and 1000 nm and is composed of a dense core from which filaments extend into the periphery. We used a combination of anti-ASC antibodies as well as much smaller nanobodies for labeling, and find that the larger antibodies reliably label the lower-density periphery whereas the nanobody, which has a lower binding affinity, is more efficient in labeling the dense core. Imaging whole cells using dSTORM, furthermore, allowed us to sort the imaged structures into a pseudo-time sequence suggesting that the endogenous ASC speck becomes denser but not much larger during its formation. The reported results add an important piece of information towards a comprehensive understanding of the supramolecular structure of the endogenous inflammasome complex. SignificanceThe assembly of ASC into a supramolecular complex referred to as the ASC speck is a critical step in the NLRP3 inflammasome response. Despite significance research, the organization and formation of the endogenous ASC speck remain elusive. We used a combination of differently sized ASC labels with super-resolution microscopy to address the organization of the ASC speck at the nanoscale. Our results allow inference of the supramolecular structure of the unperturbed, endogenous speck and characterize how it changes during ASC recruitment. We find that the complex varies in size and is composed of a dense core from which filaments extend into the periphery. Notably, our results further indicate that during inflammasome formation, ASC specks become denser but only slightly larger.

immunology↗

bioRxiv2019

Ncl1 mediated metabolic rewiring is critical during metabolic stress

Nutritional limitation has been vastly studied, however, there is limited knowledge of how cells maintain homeostasis in excess nutrients. In this study, using yeast as a model system, we show that some amino acids are toxic at higher concentrations. With cysteine as a physiologically relevant example, we delineated the pathways/processes that are altered and those that are involved in survival in presence of elevated levels of this amino acid. Using proteomics and metabolomics approach, we found that cysteine upregulates proteins involved in amino acid metabolism, alters amino acid levels, and inhibits protein translation, events that are rescued by leucine supplementation. Through a comprehensive genetic screen we show that leucine mediated effect depends on a tRNA methyltransferase (Ncl1), absence of which decouples cells transcription and translation, inhibits the conversation of leucine to ketoisocaproate and leads to TCA cycle block. We therefore, propose a role of Ncl1 in regulating metabolic homeostasis through translational control.

biochemistry↗