Biology subjects

Olieric, V.

Publications and source records attributed to Olieric, V..

6 recordsLinked to original sources

bioRxiv2026

Time-resolved functional rotation crystallography reveals protein dynamics and catalysis

Time-resolved crystallography offers a window into the transient, atomic-scale structural changes that underlie biological function. Here we introduce Time-Resolved Functional Rotation Crystallography (TR-FRX), a method that enables molecular-level studies of ligand binding and enzymatic catalysis using single protein crystals at room temperature. Unlike many state-of-the-art time-resolved methodologies, TR-FRX relies on standard rotation-based X-ray data collection and does not require serial sample delivery strategies. By dispensing nanoliter-scale droplets of ligand or substrate directly onto crystals mounted in conventional holders, TR-FRX captures real-time structural snapshots while maintaining experimental accessibility. As a proof-of-concept, we first monitor the binding of N-acetylglucosamine to hen egg-white lysozyme, demonstrating that TR-FRX can reveal ligand recognition in crystallo on sub-second timescales. We then resolve the bidirectional catalytic mechanism of a prototypical enzyme from the tricarboxylic-acid-cycle, revealing sequential cofactor and substrate binding, catalytic loop dynamics, and substrate stabilization across time points spanning from 137 milliseconds to minutes. Requiring only micrograms of protein and standard beamline infrastructure, TR-FRX provides an accessible methodology for capturing transient enzymatic states and enables 100-ms timescale studies at room temperature on single protein crystals.

biochemistry↗

bioRxiv2025

Radiation dose effects in correlative X-ray / cryo-electron microscopy of frozen hydrated biological samples

In cryo-electron microscopy (cryo-EM), imaging of biological specimens is restricted by the limited field of view and by sample thickness. Hard X-ray imaging, with its ability to penetrate samples several tens of micrometers thick, offers a complementary approach for high-resolution visualization. A major concern is whether cryo-preserved samples can withstand the handling conditions at synchrotron facilities without excessive icing, and whether the radiation exposure during X-ray imaging compromises specimen integrity, thereby hindering subsequent attempts to achieve high-resolution 3D reconstructions via cryo-EM. To evaluate this, we deposited apoferritin samples on a cryo-EM grid, exposed them to varied X-ray doses typical for X-ray tomography experiments at a synchrotron facility, and subsequently analysed the exposed particles by cryo-EM. Despite the apparent damage sustained throughout the experiment, the samples remained amenable to cryo-EM analysis, with structural details at a resolution of [~]4 [A] at the highest absorbed X-ray dose of 100 MGy. By comparison, a similar cryo-EM dataset of the apoferritin particles that were not exposed to X-rays but were mounted on the same cryo-EM grid, resulted in a 3D reconstruction at 3.17 [A] resolution. Thus, while radiation damage may limit the high-resolution information in specimens processed by cryo-X-ray tomography, the cryo-preserved biological material exposed to these high X-ray doses can be still used for subsequent cryo-EM workflows aiming to obtain structural biology insights at intermediate to high resolution. These findings lay the groundwork for an integrated imaging workflow that combines X-ray and cryo-EM techniques to enable multiscale analysis of thick vitrified biological specimens.

biophysics↗

bioRxiv2022

Anticodon-like loop-mediated dimerization in the crystal structures of HdV- like CPEB3 ribozymes

Cytoplasmic polyadenylation element-binding (CPEB) proteins are involved in many cellular processes, including cell division, synaptic plasticity, learning, and memory. A highly conserved, short mammalian ribozyme has been found within the second intron of the CPEB3 gene. Based on its cleavage mechanism and structural features, this ribozyme belongs to the hepatitis delta virus (HDV)-like ribozyme family. Here, we present the first crystallographic structures of human and chimpanzee CPEB3 ribozymes, both confirming the general topology of the HDV ribozyme with two parallel coaxial helical stacks. However, the residues involved in forming the P1.1 mini-helix, which is an integral part of the characteristic nested double pseudoknot involving P1, P2, and P3, instead participate in a seven nucleotides loop with a conformation similar to the one from the anticodon (AC) loop of tRNAs when interacting with the mRNA codon. The conformation of the loop supports the formation of a four-base pair helix by interacting with the AC-like loop from a symmetry-related ribozyme leading to ribozyme dimer formation. The present crystal structures link for the first time the sequence specificities of the CPEB3 and the HDV (genomic and antigenomic) ribozymes to their different structural features. This work corroborates the hypothesis made by Szostak that HDV ribozymes may have evolved from the CPEB3 ribozyme.

biophysics↗

bioRxiv2021

Structural insights into plasticity and discovery of remdesivir metabolite GS-441524 binding in SARS-CoV-2 macrodomain

The nsP3 macrodomain is a conserved protein interaction module that plays essential regulatory roles in host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus, targeting this protein domain may offer a therapeutic strategy to combat the current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of macrodomain crystal structures complexed with diverse naturally-occurring nucleotides, small molecules as well as nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 macrodomain structural plasticity and it provides chemical starting points for future inhibitor development.

biochemistry↗

bioRxiv2020

The inner scaffold protects from centriole fracture

Centrioles are characterized by a nine-fold arrangement of long-lived microtubule triplets that are held together by an inner protein scaffold. These structurally robust organelles experience strenuous cellular processes such as cell division or ciliary beating while performing their function. However, the molecular mechanisms underlying the stability of microtubule triplets, as well as centriole architectural integrity remain poorly understood. Here, using ultrastructure expansion microscopy (U-ExM) for nanoscale protein mapping, we reveal that POC16 and its human homolog WDR90 are components of the centriolar microtubule wall along the central core region of the centriole. We further found that WDR90 is an evolutionary microtubule associated protein with a predicted structurally homology with the ciliary inner junction protein FAP20. Finally, we demonstrate that WDR90 depletion impairs the localization of inner scaffold components, leading to centriole structural abnormalities in both human and Chlamydomonas cells. Altogether, this work highlights that POC16/WDR90 is a crucial evolutionary conserved molecular player participating in centriole architecture integrity.

cell biology↗

bioRxiv2019

The Human Telomeric Nucleosome Displays Distinct Structural and Dynamic Properties

Telomeres protect the ends of our chromosomes and are key to maintaining genomic integrity during cell division and differentiation. However, our knowledge of telomeric chromatin and nucleosome structure at the molecular level is limited. Here, we aimed to define the structure, dynamics as well as properties in solution of the human telomeric nucleosome. We first determined the 2.2 [A] crystal structure of a human telomeric nucleosome core particle (NCP) containing 145 bp DNA, which revealed the same helical path for the DNA as well as symmetric stretching in both halves of the NCP as that of the 145 bp 601 NCP. In solution, the telomeric nucleosome exhibited a less stable and a markedly more dynamic structure compared to NCPs containing DNA positioning sequences. These observations provide molecular insights into how telomeric DNA forms nucleosomes and chromatin and advance our understanding of the unique biological role of telomeres.

biochemistry↗