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 cryocrystallography has never been performed in Türkiye 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, Türkiye 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.

Rapid and efficient ambient temperature X-ray crystal 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 min with resolution extending to 2.39 Å 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.

Protocol for structure determination of SARS-CoV-2 main protease at near-physiological-temperature by serial femtosecond crystallography.

The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography. For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021).

Cooperative allostery and structural dynamics of streptavidin at cryogenic- and ambient-temperature.

Multimeric protein assemblies are abundant in nature. Streptavidin is an attractive protein that provides a paradigm system to investigate the intra- and intermolecular interactions of multimeric protein complexes. Also, it offers a versatile tool for biotechnological applications. Here, we present two apo-streptavidin structures, the first one is an ambient temperature Serial Femtosecond X-ray crystal (Apo-SFX) structure at 1.7 Å resolution and the second one is a cryogenic crystal structure (Apo-Cryo) at 1.1 Å resolution. These structures are mostly in agreement with previous structural data. Combined with computational analysis, these structures provide invaluable information about structural dynamics of apo streptavidin. Collectively, these data further reveal a novel cooperative allostery of streptavidin which binds to substrate via water molecules that provide a polar interaction network and mimics the substrate biotin which displays one of the strongest affinities found in nature.

Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing.

The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2.

Potential biomarker of circulating hsa-miR-1273g-3p level for detection of recurrent epithelial ovarian cancer.

PURPOSE: Ovarian cancer (OC) is first gynaecologic cancer that causes women death and epithelial ovarian cancer (EOC) is the most lethal ovarian cancer type. While treatment is commonly successful, some cases (10-20%) show resistance to chemotherapy which is followed by recurrence. MicroRNA (miRNA) based diagnosis methods are slightly important for recurrent ovarian cancer diagnosis. We aimed to detect novel circulating miRNAs to be used as an early diagnosis and prediction tools for recurrent EOC. METHODS: In this study, recurrent EOC serum samples and healthy control serum samples were compared for miRNA expression analysis by microarray. Microarray results were analyzed by bioinformatics tools and differentially expressed hsa-miR-1273g-3p was obtained. After microarray analysis, differentially expressed hsa-miR-1273g-3p was validated by Real-Time PCR (RT-qPCR). The relation between target genes of hsa-miR-1273g-3p and ovarian cancer were examined by Pathway Studio® (v.11.4.0.8). RESULTS: The expression of hsa-miR-1273g-3p was found to be significantly down-regulated by t test Bonferroni FWER corrected p < 0.05 and fold change > 2, in recurrence EOC compare with healthy controls groups. The RT-qPCR results confirmed that relative expressions of the serum hsa-miR-1273g-3p were significantly down-regulated in patients with recurrent EOC (p = 0.0275). Serum hsa-miR-1273g-3p levels could discriminate patients with recurrent EOC from healthy controls, with a power area under the curve (AUC) of 0.7. CONCLUSION: This study suggested that hsa-miR-1273g-3p plays a significant role in regulation of related genes, which are TNF-alfa, COL1A1, MMP-2, MMP-9, with recurrent EOC outcome. hsa-miR-1273g-3p may be used as a prognostic marker for recurrent EOC after chemotherapy.