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Several structure determination techniques such as X-ray diffraction (XRD), nuclear magnetic resonance (NMR), cryogenic electron microscopy (cryo-EM), small angle X-ray scattering (SAXS), and X-ray free electron laser (XFEL) are widely used to decode the structural information of biological macromolecules and its complexes, among which NMR, cryo-EM, SAXS, and XFEL are categorized by solution state and can only analyze the relative stereochemistry of the macromolecule, while single-crystal XRD is the ultimate method to determine the most accurate high-resolution atomic crystalline structure. The main bottle neck of X-ray crystallography is to determine the phase problem which computes the electron density to locate the actual position of the atoms. Several methods are available to solve the phase problem which includes direct methods (small molecules), molecular replacement (MR), single/multiple isomorphous replacement (SIR/MIR), and single-wavelength anomalous dispersion/multiwavelength anomalous dispersion (SAD/MAD). The scattered X-rays are recorded by charged couple device detectors and the diffraction intensity data are collected. These collected data are then processed using modern software for crystal system identification, indexing, measurement of intensities, and scaling factor to get electron density maps. Direct method is a mathematical technique used to derive the phase information directly from the observed intensities to locate the atoms. SHELXD software is used in direct methods. MR method relies upon the existence of a homologous structure from which the diffraction data are derived. MolRep, Phaser, and Balbes are some of the software used in MR technique. Also, the phase information can be fetched by isomorphous replacement method where the crystals are soaked in a heavy atom in solution and the diffraction pattern of the crystal is compared with the native. This is further classified into SIR and MIR. In order to fetch the position of the atoms in a single crystal, anomalous scattering or dispersion of heavy atoms is used, which is further classified into MAD and SAD. SOLVE/RESOLVE software are used in isomorphous replacement and anomalous dispersion. Incorporation of selenomethionine into protein during purification or by heavy atom soaking also gives a hand to solve the phase problem in X-ray crystallography. If the wavelength of the X-ray corresponds to a transition between different electron shells of the atom, there will be a modification of the phase. Moreover, synchrotron X-ray facility gives fast and precious information about the atoms with high resolution. Once the model of the structure is predicted, further refinement by computer programs like RefMac will be performed to get better quality structures. Complete protein structure can be an outbreak when the initial structures are accurately built by refining the protein structure. X-ray crystallographic structures will also provide deep insights about the orientation of individual atoms in a protein structure which unveils its function and consequently enhances drug designing.

Leaf extract of medicinally important plant Ocimum sanctum (O. sanctum) has been used for the synthesis of nickel nanoparticles (NiGs) and extraction of quercetin (Qu). Qu has been conjugated with NiGs for enhanced anticancer effect on human breast cancer MCF–7 cells. Extracted Qu was conjugated with polyethylene glycol (PEG) coated NiGs (Qu–PEG–NiGs) which was used as carriers for breast cancer treatment. Anticancer activity of Qu–PEG–NiGs was evaluated by assessing cell viability, reactive oxygen species (ROS) production, caspase activity, mitochondrial membrane potential (MMP) and changes in nuclear morphology (staining methods). 0.85 mg of quercetin was extracted from 1 g of leaves with retention time ( R t ) of 2.914 min. Loading and encapsulation efficiency of quercetin onto PEG–NiGs was 15.04% and 82% respectively and Qu–PEG–NiGs has shown a sustained release of Qu of about 84% after 48 h. Qu and Qu–PEG–NiGs showed dose dependent (1.56–50 μg/mL) anticancer effect against MCF–7 cells with IC 50 values of 50 and 6.25 μg/mL respectively which was mediated by oxidative stress due to ROS over-production that induced loss of mitochondrial membrane potential, capsase -9, -7 activities leading to apoptosis. The present study validates that Qu–PEG–NiGs can be used as a potential anticancer agent for cancer therapy.

Dengue is a mosquito-borne viral infection caused by Dengue virus (DENV) and is an emerging concern in public health affecting billions of people worldwide annually with no effective drugs available till now. Immunogenic and highly conserved properties of Non-Structural Protein 5(NS5) in DENV makes it a potent marker to identify DENV infection. DENV interfere in the innate immune signaling and thereby decreases antiviral responses and favors viral replication. Viral recognition by host pathogen recognition receptors facilitates binding of interferon (IFN) to the interferon receptors that further activates both the Signal Transducer and Activator of Transcription-2 (STAT-2) a factor producing an antiviral response. The most debilitating factor of DENV infection is emaciation of human immune system by DENV- NS5. NS5 counters the antiviral response by STAT2 degradation impeding the transcriptional activation of interferon stimulated genes through interferon stimulated response elements. The present study aims to identify inhibitors for NS5 Methyl Transferase (MTase) domain and to provide an insight into the mechanism of STAT2 degradation in the host infected with DENV. Virtual screening and molecular docking studies identified five potential inhibitors ZINC84154300, ZINC08762321, ZINC08762323, ZINC12659408 and ZINC12285470 with docking scores of -10.55, -10.53, -10.78, -11.28 and -10.78 kcal/mol respectively. To further investigate the stability of the complexes, we have used Molecular Dynamics Simulations (MD). Besides, the binding free energy of top 5 docked ligands were estimated through Molecular Mechanics Generalized Born and Surface Area Solvation (MM/GBSA) methods. This study also provides an insight on the mechanism of immunological processes involved in alleviating the antiviral immune response and computational identification of potent inhibitors for viral NS5 protein.