Biology subjects

Yapici, I.

Publications and source records attributed to Yapici, I..

4 recordsLinked to original sources

bioRxiv2023

4D Crystallography Captures Transient IF1-Ribosome Dynamics in Translation Initiation

Initiation factor 1 (IF1) is one of multiple key ligands involved in the initiation of mRNA translation, a highly dynamic and carefully-orchestrated process. However, details surrounding IF1 transient interactions with the small 30S ribosomal subunit remain incompletely understood despite characterization of unbound and fully-bound 30S states. Improvements in X-ray light sources and crystallographic techniques are now enabling time-resolved structural studies at near-physiological temperature and near-atomic resolution and thus the structural investigation of such dynamic processes. Here, we employed time-resolved serial femtosecond X-ray crystallography (TR-SFX) to probe the binding of IF1 to the small 30S ribosomal subunit in real time. Our time-resolved structural data demonstrates transient cryptic short-, mid-, and long-range allostery among different regions of the small 30 ribosomal subunit during IF1 binding, revealing small- and large-scale protein-target interactions and dynamics within intermediate macromolecular states at unprecedented temporal and spatial resolution. These data represent one of the first such 4D crystallographic studies assessing protein-protein and protein-RNA interactions and could serve as the basis for subsequent studies of the ribosome and of the multitudinous dynamic processes which underpin biology, and therefore, of life.

biophysics↗

bioRxiv2023

Ambient Temperature Bacterial Large Ribosomal Subunit Structure Enabled by Serial Femtosecond X-ray Crystallography

Ribosomes are the supramolecular complexes responsible for protein synthesis. The large 50S ribosomal subunit catalyzes the peptidyl transferase reaction and peptide bond formation between amino acids. The 50S is targeted by many known clinically effective antibiotics. Available structures, obtained at cryogenic temperatures (CT), are used for drug discovery despite that active or important target sites may display a structural configuration that is CT-induced. The introduction of ultrafast and ultrabright X-ray free electron laser (XFEL) pulses has enabled the structural observation of biological macro- and supramolecules at previously unattainable, near-physiological temperatures. In this study, we use ultrafast and ultrabright XFEL pulses to solve the apo form of 50S ribosomal subunit isolated from the extremely thermophilic bacterium Thermus thermophilus at ambient temperature (AT). The dimeric structure of the 50S subunit presented in this work is among the largest ([~]3 megadalton) structures determined using an XFEL source to date. This study demonstrates the ability to obtain new information about ribosome structural dynamics at AT through serial femtosecond X-ray crystallography (SFX). This allowed us to capture previously unobserved dynamics of ribosomal protein uL23 and coordination by hexahydrated magnesium cations at a hitherto unseen resolution at near-physiological temperature. Also, residue A2602, at the core of the peptidyl transferase center (PTC), shows a rather different orientation of the sugar moiety if compared to CT structures. In addition, our structure highlights the importance of flexible residues at both the PTC and in the binding sites for antibiotics erythromycin and chloramphenicol. The method implemented here may also serve as a starting point for future structural research involving the 50S subunit complexes by employing time-resolved mix-inject and probe kineto-crystallography experiments at XFELs. Unveiling ligand-dependent 50S dynamics at physiological temperatures shall guide further development of next-generation antibiotics that target the translation machinery.

biophysics↗

bioRxiv2022

Rapid and High Resolution Ambient Temperature Structure Determination at Turkish Light Source

High-resolution biomacromolecular structure determination is essential to better understand protein function and dynamics. Serial crystallography is an emerging structural biology technique which has fundamental limitations due to either sample volume requirements or immediate access to the competitive X-ray beamtime. Obtaining a high volume of well-diffracting, sufficient-size crystals while mitigating radiation damage remains a critical bottleneck of serial crystallography. As an alternative, we introduce the plate-reader module adapted for using a 72-well Terasaki plate for biomacromolecule structure determination at a convenience of a home X-ray source. We also present the first ambient temperature lysozyme structure determined at the Turkish Light Source (Turkish DeLight). The complete dataset was collected in 18.5 mins with resolution extending to 2.39 [A] and 100% completeness. Combined with our previous cryogenic structure (PDB ID: 7Y6A), the ambient temperature structure provides invaluable information about the structural dynamics of the lysozyme. Turkish DeLight provides robust and rapid ambient temperature biomacromolecular structure determination with limited radiation damage.

biophysics↗

bioRxiv2022

Cryogenic X-ray crystallographic studies of biomacromolecules at Turkish Light Source: Turkish DeLight

X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g. proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryo-crystallography has never been performed in Turkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, Istanbul, Turkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample for structural studies. Therefore, this article will be a valuable practical guide from sample preparation to data analysis.

biophysics↗