Biology subjects

Ourmazd, A.

Publications and source records attributed to Ourmazd, A..

8 recordsLinked to original sources

bioRxiv2022

Heterogeneity in the M. tuberculosis β-lactamase inhibition by Sulbactam

For decades, researchers have been determined to elucidate essential enzymatic functions on the atomic lengths scale by tracing atomic positions in real time. Our work builds on new possibilities unleashed by mix-and-inject serial crystallography (MISC)1-5 at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals6. Here, we report in atomic detail and with millisecond time-resolution how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating7-9, cooperativity, induced fit10,11 and conformational selection11-13 all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme non-covalently before reacting to a trans-enamine. This was made possible in part by the application of the singular value decomposition14 to the MISC data using a newly developed program that remains functional even if unit cell parameters change during the reaction.

molecular biology↗

bioRxiv2022

Energy Landscapes from Cryo-EM Snapshots: A Benchmarking Study

Biomolecules undergo complex continuous conformational motions, a subset of which are functionally relevant. Understanding, and ultimately controlling biomolecular function are predicated on the ability to map continuous conformational motions and identify the functionally relevant conformational trajectories. For equilibrium and near-equilibrium processes, the function proceeds along minimum-energy pathways on one or more energy landscapes, because higher-energy conformations are only weakly occupied. With the growing interest in identifying functional trajectories on energy landscapes, the reliable mapping of energy landscapes has become paramount. In response, various data-analytical tools for determining structural variability are emerging. A key question concerns the veracity with which each data-analytical tool can extract functionally relevant conformational trajectories from a collection of singleparticle cryo-EM snapshots. Using synthetic data as an independently known ground truth, we benchmark the ability of four leading algorithms to determine biomolecular energy landscapes and identify the functionally relevant conformational paths on these landscapes. Such benchmarking is essential for systematic progress toward atomic-level movies of continuous biomolecular function.

biophysics↗

bioRxiv2021

Energy Landscape of the SARS-CoV-2 Reveals Extensive Conformational Heterogeneity

Cryo-electron microscopy (cryo-EM) has produced a number of structural models of the SARS-CoV-2 spike, already prompting biomedical outcomes. However, these reported models and their associated electrostatic potential maps represent an unknown admixture of conformations stemming from the underlying energy landscape of the spike protein. As for any protein, some of the spikes conformational motions are expected to be biophysically relevant, but cannot be interpreted only by static models. Using experimental cryo-EM images, we present the energy landscape of the spike protein conformations, and identify molecular rearrangements along the most-likely conformational path in the vicinity of the open (so called 1RBD-up) state. The resulting global and local atomic refinements reveal larger movements than those expected by comparing the reported 1RBD-up and 1RBD-down cryo-EM models. Here we report greater degrees of "openness" in global conformations of the 1RBD-up state, not revealed in the single-model interpretations of the density maps, together with conformations that overlap with the reported models. We discover how the glycan shield contributes to the stability of these conformations along the minimum free-energy pathway. A local analysis of seven key binding pockets reveals that six out them, including those for engaging ACE2, therapeutic mini-proteins, linoleic acid, two different kinds of antibodies, and protein-glycan interaction sites, switch conformations between their known apo- and holo-conformations, even when the global spike conformation is 1RBD-up. This is reminiscent of a conformational pre-equilibrium. We found only one binding pocket, namely antibody AB-C135 to remain closed along the entire minimum free energy path, suggesting an induced fit mechanism for this enzyme.

biophysics↗

bioRxiv2020

Direct Observation of the Mechanism of Antibiotic Resistance by Mix-and-Inject at the European XFEL

In this study, we follow the diffusion and buildup of occupancy of the substrate ceftriaxone in M. tuberculosis {beta}-lactamase BlaC microcrystals by structural analysis of the enzyme substrate complex at single millisecond time resolution. We also show the binding and the reaction of an inhibitor, sulbactam, on a slower millisecond time scale. We use the mix-and-inject technique to initiate these reactions by diffusion, and determine the resulting structures by serial crystallography using ultrafast, intense X-ray pulses from the European XFEL (EuXFEL) arriving at MHz repetition rates. Here, we show how to use the EuXFEL pulse structure to dramatically increase the size of the data set and thereby the quality and time resolution of "molecular movies" which unravel ligand binding and enzymatically catalyzed reactions. This shows the great potential for the EuXFEL as a tool for biomedically relevant research, particularly, as shown here, for investigating bacterial antibiotic resistance. One Sentence SummaryDirect observation of fast ligand binding in a biomedically relevant enzyme at near atomic resolution with MHz X-ray pulses at the European XFEL.

biophysics↗

bioRxiv2020

Single-femtosecond atomic-resolution observation of a protein traversing a conical intersection

The structural dynamics of a molecule are determined by the underlying potential energy landscape. Conical intersections are funnels connecting otherwise separate energy surfaces. Posited almost a century ago 1, conical intersections remain the subject of intense scientific investigation 2-4. In biology, they play a pivotal role in vision, photosynthesis, and DNA stability 5,6. In ultrafast radiationless de-excitation 1,7, they are vital to ameliorating photon-induced damage. In chemistry, they tightly couple the normally separable nuclear and electronic degrees of freedom, precluding the Born-Oppenheimer approximation 8. In physics, they manifest a Berry phase, giving rise to destructive interference between clockwise and anti-clockwise trajectories around the conical intersection 9. Accurate theoretical methods for examining conical intersections are at present limited to small molecules. Experimental investigations are challenged by the required time resolution and sensitivity. Current structure-dynamical understanding of conical intersections is thus limited to simple molecules with around 10 atoms, on timescales of about 100 fs or longer 10. Spectroscopy can achieve better time resolution, but provides only indirect structural information. Here, we present single-femtosecond, atomic-resolution movies of a 2,000-atom protein passing through a conical intersection. These movies, extracted from experimental data by geometric machine learning, reveal the dynamical trajectories of de-excitation via a conical intersection, yield the key parameters of the conical intersection controlling the de-excitation process, and elucidate the topography of the electronic potential energy surfaces involved.

biophysics↗

bioRxiv2018

Functional Pathways of Biomolecules Retrieved from Single-particle Snapshots

We present a new approach to determining the conformational changes associated with biological function, and demonstrate its capabilities in the context of experimental single-particle cryo-EM snapshots of ryanodine receptor (RyR1), a Ca2+-channel involved in skeletal muscle excitation/contraction coupling. These results include the detailed conformational motions associated with functional paths including transitions between energy landscapes. The functional motions differ substantially from those inferred from discrete structures, shedding new light on the gating mechanism in RyR1. The differences include the conformationally active structural domains, the nature, sequence, and extent of conformational motions involved in function, and the way allosteric signals are transduced within and between domains. The approach is general, and applicable to a wide range of systems and processes.

biophysics↗

bioRxiv2017

Authentic Enzyme Intermediates Captured "on-the-fly" by Mix-and-Inject Serial Crystallography

Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Here, we demonstrate a general method for capturing enzyme catalysis in-action by mix-and-inject serial crystallography. Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis -lactamase with the 3rd generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation on the millisecond to second time scales including the crossover from transition state kinetics to steady-state kinetics.\n\nSynopsisAn enzymatically catalyzed reaction is initiated by diffusion based mixing of substrate and followed at runtime by time-resolved serial crystallography using a free electron laser.

biophysics↗

bioRxiv2017

Conformational Dynamics and Energy Landscapes of Ligand Binding in RyR1

Using experimental single-particle cryo-EM snapshots of ryanodine receptor (RyR1), a Ca2+-channel involved in skeletal muscle excitation/contraction coupling, we present quantitative free-energy landscapes, reaction coordinates, and three-dimensional movies of the continuous conformational changes associated with the binding of activating ligands. Our results show multiple routes to ligand binding with comparable branching ratios. All high-probability routes involve significant conformational changes before and after the binding of ligands. We also present new insights into the local structural changes along the ligand-binding route, including accommodations at the calcium, ATP, and caffeine binding sites. These observations shed new light on the mechanisms and conformational routes to ligand binding.

biophysics↗