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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, İstanbul, 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., NSF Science and Technology Center Grant; Biology with X-ray Lasers, BioXFEL; Scientific and Technological Research Council of Turkey (TÜBİTAK); TÜBİTAK 2218 - National Postdoctoral Research Fellowship Program; TÜBİTAK 2232 International Outstanding Researchers Program; 2232 International Fellowship for Outstanding Researchers Program; European Union (EU); Horizon 2020; 2236 CoCirculation2 Program; TÜBİTAK 1001 Scientific and Technological Research Projects Funding Program

Photodynamic therapy (PDT) is a photo chemotherapeutic strategy that is the application of photosensitizing agent and light on disease or tumor site. The aim of this study is to confirm the feasibility for femtosecond (fs) laser for aminolevulinate (ALA) mediated PDT on skin, breast and bladder cancer cells. Also the remarkable aspects of ALA mediated and laser induced PDT with respect to other literally known applications were investigated. Metastatic melanoma cells SK-MEL30, mammary epithelial carcinoma cells MCF-7 and bladder cancer cells UMUC-3 were treated with ALA and then the cells were irradiated by fs laser at thirty wavelengths in between 230 and 800 nm for 30s and 60s. Anti-cancer effects of ALA phototherapy on different cancer cell lines were determined. Protoporphyrin IX (PpIX) accumulation was visualized by confocal microscopy. The effective PDT wavelengths were applied to evaluate the degree of apoptosis and necrosis in cells. The viability tests demonstrated that wavelengths 400-440 nm and 600-630 nm were found to decrease the viability on three model cell lines. PDT at 630 nm exerted cell death by necrosis and apoptosis after 30 s and 60 s periods. This paper confirms that ALA and femtosecond laser mediated PDT may be used together as therapeutic and diagnostic method to target breast, skin and urinary bladder cancer cells. The use of fs laser allows the flexibility for optimization of wavelength for photosensitizing agents., Scientific and Technological Research Council of Turkey (TÜBİTAK)

The HIV-1 Gag protein binds to the host cell membrane and assembles into immature particles. Then, in the course of immature virion budding, activated protease cleaves Gag into its main components: MA, CA, NC, and p6 proteins. The highly basic residues of MA predominantly interact with the acidic head of phosphatidyl-inositol-4,5-bisphosphate (PI(4,5)P2) inserted into the membrane. Our research group developed L-Heptanoylphosphatidyl Inositol Pentakisphosphate (L-HIPPO) and previously confirmed that this compound bound to the MA more strongly than PI(4,5)P2 and inositol hexakisphosphate (IP6) did. Therefore, herein we rationally designed eight new L-HIPPO derivatives based on the fact that the most changeable parts of L-HIPPO were two acyl chains. After that, we employed molecular docking for eight compounds via Maestro software using high-resolution crystal structures of MA in complex with IP6 (PDB IDs: 7E1I, 7E1J, and 7E1K), which were recently elucidated by our research group. The most promising docking scores were obtained with benzene-inserted compounds. Thus, we generated a library containing 213 new aromatic groupinserted L-HIPPO derivatives and performed the same molecular docking procedure. According to the results, we determined the nine new L-HIPPO derivatives most effectively binding to the MA with the most favorable scoring functions and pharmacokinetic properties for further exploration., Scientific and Technological Research Council of Turkey (TÜBİTAK); European Union (EU); Horizon 2020; Marie Sklodowska-Curie Actions Cofund program; European Commission; Co-Funded Brain Circulation2 Scheme; 2236 CoCirculation2 Program; European Research Council (ERC); Istanbul University

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., National Science Foundation (NSF) Science and Technology Centers; BioXFEL; National Institutes of Health (NIH); National Institute of General Medical Sciences (NIGMS); Scientific and Technological Research Council of Turkey (TÜBİTAK); 2232 International Fellowship for Outstanding Researchers Program of TÜBİTAK; 2244 Industrial PhD Program of TÜBİTAK; 1001 TÜBİTAK Supporting Scientific and Technological Research Projects Program; Linac Coherent Light Source (LCLS); Stanford Synchrotron Light Source (SSRL); SLAC National Accelerator Laboratory; U.S. Department of Energy, Office of Science, Basic Energy Sciences

Ayan et al. report two structures of the protein streptavidin - one at ambient temperature determined using serial femtosecond crystallography and a second one determined at cryogenic temperature. These results provide insights into the structural dynamics of apo streptavidin and reveal a cooperative allostery between monomers for binding to biotin, and the findings are supported by GNM analysis. 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 angstrom resolution and the second one is a cryogenic crystal structure (Apo-Cryo) at 1.1 angstrom 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., National Science Foundation (NSF) Science and Technology Centers; 2232 International Fellowship for Outstanding Researchers Program; 1001 Scientific and Technological Research Projects Funding Program; Scientific and Technological Research Council of Turkey (TÜBİTAK); U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences (OBES); National Institutes of Health, National Institute of General Medical Sciences (NIGMS)

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.

Oligomerization of Pr55(Gag) is a critical step of the late stage of the HIV life cycle. It has been known that the binding of IP6, an abundant endogenous cyclitol molecule at the MA domain, has been linked to the oligomerization of Pr55(Gag). However, the exact binding site of IP6 on MA remains unknown and the structural details of this interaction are missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and GNM-based transfer entropy calculations identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the binding of Pr55(Gag) to membrane. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18-33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles' membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves IP6 coordination of the oligomerization of outer MA and inner CA domain's 2D layers during assembly and budding.

The ammonia-oxidizing thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle is one of the most energy-efficient CO2 fixation cycles discovered thus far. The protein encoded by Nmar_1308 (from Nitrosopumilus maritimus SCM1) is a promiscuous enzyme that catalyzes two essential reactions within the thaumarchaeal 3HP/4HB cycle, functioning as both a crotonyl-CoA hydratase (CCAH) and 3-hydroxypropionyl-CoA dehydratase (3HPD). In performing both hydratase and dehydratase activities, Nmar_1308 reduces the total number of enzymes necessary for CO2 fixation in Thaumarchaeota, reducing the overall cost for biosynthesis. Here, we present the first high-resolution crystal structure of this bifunctional enzyme with key catalytic residues in the thaumarchaeal 3HP/4HB pathway., National Science Foundation (NSF) Science and Technology Centers; Science and Technology Centers; Scientifc and Technological Research Council of Turkey (TÜBİTAK); US Department of Energy Joint Genome Institute

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., National Science Foundation (NSF) Science and Technology Centers; BioXFEL; Scientific and Technological Research Council of Turkey (TÜBİTAK); Bahçeşehir University (BAU) Scientific Research Projects (BAP)

Since early 2020, COVID-19 has grown to affect the lives of billions globally. A worldwide investigation has been ongoing for characterizing the virus and also for finding an effective drug and developing vaccines. As time has been of the essence, a crucial part of this research has been drug repurposing; therefore, confirmation of in silico drug screening studies have been carried out for this purpose. Here we demonstrated the possibility of screening a variety of drugs efficiently by leveraging a high data collection rate of 120 images/second with the new low-noise, high dynamic range ePix10k2M Pixel Array Detector installed at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS). The X-ray Free-Electron Laser (XFEL) is used for remote high-throughput data collection for drug repurposing of the main protease (Mpro) of SARS-CoV-2 at ambient temperature with mitigated X-ray radiation damage. We obtained multiple structures soaked with nine drug candidate molecules in two crystal forms. Although our drug binding attempts failed, we successfully established a high-throughput Serial Femtosecond X-ray crystallographic (SFX) data collection protocol., National Science Foundation (NSF) Science and Technology Centers Grant; Biology with X-ray Lasers (BioXFEL); Scientific and Technological Research Council of Turkey (TÜBİTAK); 2232 International Fellowship for Outstanding Researchers Program; 2244 Industrial PhD Fellowship Program; 1001 Scientific and Technological Research Projects Funding Program

Oligomerization of Pr55(Gag) is a critical step of the late stage of the HIV life cycle. It has been known that the binding of IP6, an abundant endogenous cyclitol molecule at the MA domain, has been linked to the oligomerization of Pr55(Gag). However, the exact binding site of IP6 on MA remains unknown and the structural details of this interaction are missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and GNM-based transfer entropy calculations identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the binding of Pr55(Gag) to membrane. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18-33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles' membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves IP6 coordination of the oligomerization of outer MA and inner CA domain's 2D layers during assembly and budding., National Science Foundation (NSF) Science and Technology Centers Grant; BioXFEL; Scientific and Technological Research Council of Turkey (TÜBİTAK); 2232 International Fellowship for Outstanding Researchers Program; Grant-in-Aid for Scientific Research; Grant-in-Aid for Research Activity Start-up; Japanese Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research; Grant-in and Aid for JSPS Research Fellow; Japan Agency for Medical Research and Development Basis for Supporting Innovative Drug Discovery and Life Science Research; Platform for Drug Discovery, Informatics, and Structural Life Science (MEXT); Japan Agency for Medical Research and Development (AMED)