Biology subjects

Ciftci, H. I.

Publications and source records attributed to Ciftci, H. I..

4 recordsLinked to original sources

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↗

bioRxiv2019

Coupled inter-subunit dynamics enable the fastest CO2-fixation by reductive carboxylases

Enoyl-CoA carboxylases/reductases (ECRs) belong to the most efficient CO2-fixing enzymes described to date. However, the molecular mechanisms underlying ECRs extraordinary catalytic activity on the level of the protein assembly remain elusive. Here we used a combination of ambient temperature X-ray Free Electron Laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from Kitasatospora setae. K. setae ECR is a homo-tetramer that differentiates into a dimer of dimers of open- and closed-form subunits in the catalytically active state. Using molecular dynamics simulations and structure-based mutagenesis, we show that catalysis is synchronized in K. setae ECR across the pair of two dimers. This conformational coupling of catalytic domains is conferred by individual amino acids to achieve high CO2-fixation rates. Our results provide unprecedented insights into the dynamic organization and synchronized inter- and intra-subunit communications of this remarkably efficient CO2-fixing enzyme during catalysis. Significance StatementFixation of CO2 offers real potential for reaching negative CO2 emissions in bioenergy, and bioproduct utilization. The capture and conversion of atmospheric CO2 remains a challenging task. Existing biological systems can be exploited and optimized for this use. Bacterial enoyl-CoA carboxylases/reductases (ECRs) encompass the fastest CO2-fixing enzymes found in nature to date. However, the mechanisms underlying ECRs extraordinary catalytic activity remain elusive. Our structural, computational, and biochemical results elucidate the dynamic structural organization of the ECR complex and describe how coupled motions of catalytic domains in the ECR tetramer drive carboxylation. This mechanistic understanding is critical for engineering highly efficient CO2-fixing biocatalysts for bioenergy and bioproduct applications.

biophysics↗

bioRxiv2019

Serial femtosecond X-ray diffraction of HIV-1 Gag MA-IP6 microcrystals at ambient temperature

The Human immunodeficiency virus-1 (HIV-1) matrix (MA) domain is involved in the highly regulated assembly process of the virus particles that occur at the host cells plasma membrane. High-resolution structures of the MA domain determined using cryo X- ray crystallography have provided initial insights into the possible steps in the viral assembly process. However, these structural studies have relied on large and frozen crystals in order to reduce radiation damage caused by the intense X-rays. Here, we report the first XFEL study of the HIV-1 MA domains interaction with inositol hexaphosphate (IP6), a phospholipid headgroup mimic. We also describe the purification, characterization and microcrystallization of two MA crystal forms obtained in the presence of IP6. In addition, we describe the capabilities of serial femtosecond X-ray crystallography (SFX) using X-ray free-electron laser (XFEL) to elucidate the diffraction data of MA-IP6 complex microcrystals in liquid suspension at ambient temperature. Two different microcrystal forms of MA-IP6 complex both diffracted to beyond 3.5 [A] resolution, demonstrating the feasibility of using SFX to study the complexes of MA domain of HIV-1 Gag polyprotein with IP6 at near-physiological temperatures. Further optimization of the experimental and data analysis procedures will lead to better understanding of the MA domain of HIV-1 Gag and IP6 interaction at high resolution and provide basis for optimization of the lead compounds for efficient inhibition of the Gag protein recruitment to the plasma membrane prior to virion formation.

biophysics↗

bioRxiv2018

Structure of the 30S ribosomal decoding complex at ambient temperature

The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New lightsources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 [A] resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation.

biophysics↗