Biology subjects

Marquez, J. A.

Publications and source records attributed to Marquez, J. A..

6 recordsLinked to original sources

bioRxiv2025

AXIS: A Lab-in-the-Loop Machine Learning approach for generalized detection of macromolecular crystals.

Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design. Nowadays, the use of robotic systems for crystal growth and diffraction analysis is widespread and high throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field. However, the identification of crystals is still largely carried out through manual inspection, sometimes involving tens of thousands of images, which represents a bottleneck in an otherwise highly automated process. Here we describe AXIS, an AI-based Crystal Identification System combining the DINOv2 computer vision model, state-of-the-art transfer learning and MARCO, the largest crystallization dataset available to date, for automated crystal detection. AXIS can operate both with visible and UV light images and integrates a Lab-In-The-Loop approach combining ML and expert inputs for continuous learning and specialization. AXIS enables automated annotation of large crystallization image datasets with performance and accuracy comparable to that of human experts and the Lab-In-The-Loop approach introduced here enables efficient adaptation to local conditions facilitating widespread application, which has been a major limitation to date. AXIS can help correct human errors in image annotation and removes critical bottlenecks, particularly in the context of extensive crystallization screens or high throughput applications like fragment and ligand screening unlocking the potential for higher levels of automation that are key both in fundamental and translational research. Appendix A. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/685844v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@c47ab4org.highwire.dtl.DTLVardef@153ce2dorg.highwire.dtl.DTLVardef@1050086org.highwire.dtl.DTLVardef@165965b_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

EnsembleFlex: Protein Structure Ensemble Analysis Made Easy

EnsembleFlex is a novel tool designed to perform dual-scale flexibility analysis of protein structure ensembles, encompassing both backbone and side-chain dynamics. It integrates dedicated superposition methodologies, two dimension reduction techniques, customizable clustering, an automated ligand binding site analysis and analysis of conserved water molecules, enhancing the accuracy of ensemble flexibility analysis and visualisation. With a user-friendly, no-coding graphical interface as well as a code-based option, EnsembleFlex is accessible to a broad range of researchers. The tool complements experimental ensemble data analysis with predictive methods based on elastic network models, bridging the gap between experimental and computational approaches in protein flexibility analysis, and does so from a global backbone-based perspective, as well as from a focused side-chain and binding site-centred point of view. The need for reliable, streamlined, fast, and user-friendly tools for structural ensemble analysis is critical in todays research environment, and EnsembleFlex addresses these needs efficiently, offering an accessible solution that integrates powerful analysis methods in a single package. Its wide applicability across various domains of molecular design underscores the importance of streamlined flexibility analysis in scientific research.

molecular biology↗

bioRxiv2023

X-Ray Structure and enzymatic study of a Bacterial NADPHoxidase highlight the activation mechanism of eukaryoticNOX

NADPH oxidases (NOX) are transmembrane proteins that professionally produce reactive oxygen species (ROS) and are distributed widely in both eukaryotes and prokaryotes. Eukaryotes use the ROS products for innate immune defense and signaling; the seven human isoforms of NOX participate in critical physiological and pathophysiological processes. Recently solved structures of two human NOX isoforms provide much new information, but do not fully elucidate controls on the electron transfer pathway from NAD(P)H substrate through FAD and heme cofactors to the final ROS product. SpNOX, a bacterial NOX homolog from Streptococcus pneumoniae, shows robust constitutive activity in detergent solution, making it a good prototype for exploring electron transfer in the NOX family. Here we present crystal structures of wildtype and mutant full-length and dehydrogenase (DH) domain-only constructs of SpNOX. The isolated DH domain acts as a flavin reductase, and both DH and full-length constructs use either NADPH or NADH as substrate. Our data supports hydride transfer from NAD(P)H to FAD as the rate limiting step in electron transfer. Using the DH domain, we demonstrate the role of F397 in allowing access of nicotinamide to the flavin isoalloxazine, while in the full-length construct, we used mutants and flavin analogs to confirm the contribution of both domains to flavin binding observed in the structure. Comparison with homologous enzymes suggests distal heme access may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity of a given NOX family member. Based on this comparison, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2s requirement for activation.

biochemistry↗

bioRxiv2023

Plasticity of the binding pocket in peptide transporters underpins promiscuous substrate recognition

Proton-coupled oligopeptide transporters (POTs) are promiscuous transporters of the Major Facilitator Superfamily, that constitute the main route of entry for a wide range of dietary peptides and orally administrated peptidomimetic drugs. Given their clinical and pathophysiological relevance, several bacterial and mammalian POT homologs have been extensively studied on a structural and molecular level. However, the molecular basis of recognition and transport of the wide range of peptide substrates has remained elusive. Here we present 14 X-ray structures of the bacterial POT DtpB in complex with chemically diverse di- and tripeptides, providing novel insights into the plasticity of the conserved central binding cavity. We analyzed binding affinities for more than 80 peptides and monitored uptake by a fluorescence-based transport assay. To probe if all natural 8400 di- and tripeptides can bind to DtpB, we employed state-of-the-art molecular docking and machine learning and conclude that peptides of a specific subset with compact hydrophobic residues are the best DtpB binders.

biophysics↗

bioRxiv2021

An automated platform for structural analysis of membrane proteins through serial crystallography

Membrane proteins are central to many pathophysiological processes yet remain very difficult to analyze at a structural level. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins due to lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperature and in a single support. We apply this approach to two human integral membrane proteins, which allowed us to capture different conformational states of intramembrane enzyme-product complexes and analyze the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting and serial crystallography enabling screening of small molecule libraries with membrane protein crystals grown in meso. This approach brings badly needed automation for this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins. HighlightsO_LIA fully automated, online workflow enables rapid determination of membrane protein structures by serial X-ray crystallography (SSX). C_LIO_LIHigh resolution room temperature and cryogenic structures of ADIPOR2 provide insights into the dynamic nature of receptor:ligand interactions. C_LIO_LIA web-based application allows remote user-guided experimental design and execution. C_LIO_LIAn automated SSX-based ligand discovery pipeline for integral membrane proteins is introduced. C_LI

biochemistry↗

bioRxiv2019

Unveiling the dimer/monomer propensities of Smad MH1-DNA complexes

R-Smads are effectors of the transforming growth factor {beta} (TGF{beta}) superfamily and along with Smad4 form trimers to interact with other transcription factors and with DNA. The 5GC-DNA complexes determined here by X-ray crystallography for Smad5 and Smad8 proteins corroborate that all MH1 domains bind SBE and 5GC sites similarly, although Smad2/3/4 MH1 domains bind DNA as monomers whereas Smad1/5/8 form helix-swapped dimers. To examine the relevance of the dimerization phenomenon and to exclude a possible crystallography-induced dimeric state, we studied these MH1 domains in solution. The results show that Smad5/8 domains populate dimers and monomers in equilibrium, whereas their Smad2/3/4 counterparts adopt monomeric conformations. We also found that swapping the loop1 sequence between Smad5 and Smad3 results in the Smad5 chimera-DNA complex crystallizing as a monomer, revealing that the loop1 sequence determines the monomer/dimer propensity of Smad MH1-domains. We propose that the distinct MH1-dimerization status of TGF{beta} and BMP activated Smads influences the interaction with specific loci genome-wide by distinct R-Smad and Smad4 complexes. SignificanceTGF{beta}- and BMP-activated R-Smads were believed to have different preferences with respect to the recognition of DNA motifs and to respond to specific activation inputs. However, recent results indicate that several types of R-Smads can be activated by similar receptors and that all Smads might recognize various DNA motifs. These results pose new questions as to why different types of R-Smads have been conserved for more than 500 million years if they could have a redundant function. They also raise questions as to how different Smad complexes recognize specific clusters of DNA motifs genome-wide. Here, using structural biology approaches, we elucidate some of the rules that help define the dimers of Smad-DNA complexes and propose how dimers and monomers could influence the composition of Smad complexes, as well as the recognition of specific cis-regulatory elements genome-wide. HighlightsR-Smads and Smad4 interact with 5GC and GTCT sites using a conserved binding mode. Functional differences of TGF{beta}- and BMP-activated R-Smads are not exclusively related to DNA specificity. Dimer/monomer propensities are detected in solution and in the absence of DNA. Loop1 sequence determines the propensity of R-Smads to form monomers or dimers in complexes with DNA. Author ContributionsL.R., Z.K., and T.G. designed and performed most experiments and coordinated collaborations with other authors. L.R. and E.A. cloned, expressed and purified all proteins, L.R., R.F., C.T., N.M., and J.C performed EMSA experiments. L.R., E.A., T.G., T.C., P.M.M., and M.J.M. performed the SAXS and NMR measurements and analyzed the data. P.M.M. analyzed the clustering of DNA motifs in ChIP-Seq data. Z.K. B.B. and R.P. screened crystallization conditions, collected X-ray data and determined the structures. Z.K., R.F. R.P., T.G., J.A.M., and M.J.M. analyzed the structures. All authors contributed ideas to the project. M.J.M. and R.P. supervised the project. M.J.M. wrote the manuscript with contributions from all other authors. The authors declare no conflict of interest. Data DepositionNMR assignments and chemical shifts have been deposited in the Biological Magnetic Resonance Data Bank, BMRB entry 27548, and the Small-angle scattering data and models have been deposited in SASBDB, entries SASDE32 (Smad5) and SASDE42 (Smad8). Densities and coordinates have been deposited in the Protein Data Bank, entries 6FZS (Smad5), 6FZT (Smad8), 6TBZ (Smad5_3 chimera), 6TCE (Smad5_gly mutant). This article contains supporting information.

molecular biology↗