Biology subjects

Orlans, J.

Publications and source records attributed to Orlans, J..

7 recordsLinked to original sources

bioRxiv2025

A user-friendly goniometer-compatible fixed-target platform formacromolecular crystallography at synchrotrons

Fixed-target platforms provide convenient support for microcrystals during serial X-ray crystallography studies using synchrotron radiation. Here, we describe a simple, user-friendly 3D-printed support where the crystals are sandwiched between two layers of thin X-ray transparent membrane resulting in very low scattering background. The platform is compatible with magnetic mounting onto the standard goniometer of macromolecular crystallography beamlines. Our design utilizes a 96-well frame that facilitates hanging-drop experiments directly on the membrane using conventional crystallization plates, thereby eliminating multiple pipetting and crystal handling steps. Crystals can be enclosed into a sandwich and packed into cassettes, preventing the risk of the sample drying out during room-temperature transportation to synchrotron sources. The versatility of the platform is demonstrated by five structures solved using different crystallization- and data-collection strategies. Single crystal rotational-crystallography at both room- and cryogenic-temperatures using large crystals of lysozyme is shown. On-chip microcrystallization is illustrated by use of a photosynthetic reaction center as an example. Finally, serial crystallography data collection at room-temperature from microcrystals of photosynthetic reaction center as well as cytochrome c oxidase crystallized in lipidic cubic phase is presented.

biophysics↗

bioRxiv2025

Novel polymer fixed-target microfluidic platforms with an ultra-thin moisture barrier for serial macromolecular crystallography

The advent of ultrabright fourth generation X-ray light sources, including X-ray free-electron lasers (XFELs) and diffraction limited synchrotrons, has significantly advanced the field of serial macromolecular protein crystallography (SX). SX experiments demand a continuous supply of fresh microcrystalline sample, ideally while minimizing overall sample consumption. Here, we introduce a novel, robust, and user-friendly polymer film technology that can be assembled in various configurations to encapsulate protein microcrystals and provide sample support for SX. This system provides an efficient hydration barrier over extended durations while maintaining an exceptionally low X-ray background. We have validated this technology by assessing hydration retention under both ambient and ultra-high vacuum conditions, and by evaluating its mechanical stability under XFEL pulses. Furthermore, we have demonstrated the effectiveness of this approach in two room-temperature serial crystallography studies to determine the structure of a 24 kDa Rapid Encystment Phenotype (REP24) protein from Franciscella tularensis.

biophysics↗

bioRxiv2025

Combining MicroED and native mass spectrometry for structural discovery of enzyme-biosynthetic inhibitor complexes

With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach resolves structures of the epoxide-based cysteine protease inhibitor, and natural product, E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. The combined structural power of MicroED and the analytical capabilities of native mass spectrometry (ED-MS) allows assignment of papain structures bound to E-64-like ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors, and crude biosynthetic reactions. ED-MS further discriminates the highest-affinity ligand soaked into microcrystals from a broad inhibitor cocktail, and identifies multiple similarly high-affinity ligands soaked into microcrystals simultaneously. This extends to libraries of printed ligands dispensed directly onto TEM grids and later soaked with papain microcrystal slurries. ED-MS identifies papain binding to its preferred natural products, by showing that two analogues of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analogue from crude biosynthetic reactions, without purification. This illustrates the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets.

biophysics↗

bioRxiv2025

Ambient temperature structural studies of Penicillin-binding Protein 2a of Methicillin-Resistant Staphylococcus aureus with XFELs and synchrotrons

Penicillin-binding protein 2a (PBP2a) is a transpeptidase responsible for the {beta}-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA), posing significant challenges to antibiotic therapy. PBP2as unique structural features, including its highly flexible active site and allosteric regulation, enable it to maintain catalytic activity even in the presence of {beta}-lactam antibiotics. Despite extensive characterization using cryogenic crystallography, key questions remain about its dynamic properties and conformational changes under near-physiological conditions. Room-temperature crystallography methods, particularly serial femtosecond X-ray crystallography (SFX) at XFELs, provide a powerful approach to capture these dynamics. Unlike cryogenic conditions that can constrain protein flexibility, SFX enables the study of conformational variability and interaction networks closer to physiological states. Here, we present the first room-temperature structures of PBP2a obtained using SFX to uncover insights into the enzymes flexibility, allosteric communication, and catalytic mechanisms. These findings are built upon optimized large-scale production and crystallization protocols for PBP2a, ensuring high-quality microcrystals suitable for SFX data collection. The room-temperature structures reveal novel interaction patterns, including unique salt bridge networks and dynamic structural elements absent in cryogenic studies. Furthermore, comparative analyses highlight how environmental conditions influence the conformational states of PBP2a, providing new perspectives on its resistance mechanisms. By integrating structural data from EuXFEL and LCLS, this study not only enhances our understanding of PBP2as functional dynamics but also underscores the value of room-temperature crystallography in studying antibiotic resistance. These insights could guide the design of next-generation {beta}-lactam antibiotics capable of overcoming PBP2a-mediated resistance.

biochemistry↗

bioRxiv2024

The nucleating agent crystallophore induces instant protein crystallization

The rapid preparation of homogeneous suspensions of micro- or nano-crystals is a crucial step in serial crystallography. We show how additives, such as the crystallophore (TbXo4) that acts as a molecular glue by promoting protein-protein interactions, can facilitate sample preparation for both serial synchrotron crystallography (SSX) and micro electron diffraction (3D ED). This lanthanide complex was used here for its nucleating properties to crystallize hen egg white lysozyme. SAXS monitoring indicates that crystals formed in a few minutes in low salt conditions that would not lead to spontaneous nucleation. Resulting micro- and nano-crystals were successfully used to determine the structure of the lysozyme-TbXo4 complex by SSX and 3D ED, illustrating the diffraction quality of the produced crystals and the usefulness of such compounds in the sample preparation pipeline for serial crystallography.

biophysics↗

bioRxiv2024

Droplet microfluidics for time-resolved serial crystallography

Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here we report the use of droplet miniaturisation for the controlled production of uniform crystals, providing an avenue for controlled diffusion and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3-{micro}m lysozyme crystals and 2-{micro}m crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2 milliseconds were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be used to advance time-resolved serial crystallography.

biochemistry↗

bioRxiv2023

Structural dynamics and functional cooperativity of human NQO1 by ambient temperature serial crystallography and simulations

The human NQO1 (hNQO1) is a FAD-dependent oxidoreductase that catalyzes the two-electron reduction of quinones to hydroquinones, being essential for the antioxidant defense system, stabilization of tumor suppressors, and activation of quinone-based chemotherapeutics, and it is over-expressed in several tumors, which makes it an attractive cancer drug target. To decipher new structural insights into the flavin reductive half-reaction of the catalytic mechanism of hNQO1, we have carried serial crystallography experiments at new ID29 beamline of the ESRF to determine, to the best of our knowledge, the first structure of the hNQO1 in complex with NADH. The use of room temperature serial crystallography with microcrystals has been key to study this mechanism. We have also performed molecular dynamics simulations of free hNQO1 and in complex with NADH. Both structural results and MD simulations have supported that the binding of NADH significantly decreases protein dynamics and stabilizes hNQO1 especially at the dimer core and interface. This is the first structural evidence that the hNQO1 functional cooperativity is driven by structural communication between the active sites through long-range propagation of cooperative effects across the hNQO1 structure. Altogether, these results pave the way for future time-resolved studies, both at XFELs and synchrotrons, of the dynamics of hNQO1 upon binding to NADH as well as during the FAD cofactor reductive half-reaction. This knowledge will allow us to reveal unprecedented structural information of the relevance of the dynamics during the catalytic function of hNQO1.

biochemistry↗