The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against ...COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M
), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M
In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.
Reperfusion injury is still a major challenge that impedes neuronal survival in ischemic stroke. However, the current clinical treatments are remained on single pathological process, which are due to ...lack of comprehensive neuroprotective effects. Herein, a macrophage‐disguised honeycomb manganese dioxide (MnO2) nanosphere loaded with fingolimod (FTY) is developed to salvage the ischemic penumbra. In particular, the biomimetic nanoparticles can accumulate actively in the damaged brain via macrophage‐membrane protein‐mediated recognition with cell adhesion molecules that are overexpressed on the damaged vascular endothelium. MnO2 nanosphere can consume excess hydrogen peroxide (H2O2) and convert it into desiderated oxygen (O2), and can be decomposed in acidic lysosome for cargo release, so as to reduce oxidative stress and promote the transition of M1 microglia to M2 type, eventually reversing the proinflammatory microenvironment and reinforcing the survival of damaged neuron. This biomimetic nanomedicine raises new strategy for multitargeted combined treatment of ischemic stroke.
Macrophage‐disguised FTY‐loaded MnO2 nanoparticles (Ma@(MnO2+FTY)) are engineered to salvage the ischemic penumbra. Nanoparticles can accumulate actively in the ischemic region to protect neurons directly via consuming ROS and generating O2. In addition, the nanoparticles can also reverse the proinflammatory microenvironment by promoting the phenotypic transition of microglia through multiple signaling pathways, increasing the protection effects on damaged neurons.
Serial crystallography is a powerful technique in structure determination using many small crystals at X‐ray free‐electron laser or synchrotron radiation facilities. The large diffraction data ...volumes require high‐throughput software to preprocess the raw images for subsequent analysis. ClickX is a program designated for serial crystallography data preprocessing, capable of rapid data sorting for online feedback and peak‐finding refinement by parameter optimization. The graphical user interface (GUI) provides convenient access to various operations such as pattern visualization, statistics plotting and parameter tuning. A batch job module is implemented to facilitate large‐data‐volume processing. A two‐step geometry calibration for single‐panel detectors is also integrated into the GUI, where the beam center and detector tilting angles are optimized using an ellipse center shifting method first, then all six parameters, including the photon energy and detector distance, are refined together using a residual minimization method. Implemented in Python, ClickX has good portability and extensibility, so that it can be installed, configured and used on any computing platform that provides a Python interface or common data file format. ClickX has been tested in online analysis at the Pohang Accelerator Laboratory X‐ray Free‐Electron Laser, Korea, and the Linac Coherent Light Source, USA. It has also been applied in post‐experimental data analysis. The source code is available via https://github.com/LiuLab‐CSRC/ClickX under a GNU General Public License.
A Python‐based program for serial crystallography experimental data preprocessing is developed for both online and offline analysis. Enhanced features include a graphical user interface, batch job execution and fast parameter optimizations.
•MicroED and SFX are capable of determining structures of proteins from nano/microcrystals.•The MicroED and SFX techniques are highly complementary.•SFX provides significant advantages for ...time-resolved studies.•MicroED produces structures from a very small number of microcrystals.
In recent years, nano and microcrystals have emerged as a valuable source of high-resolution structural information owing to the invention of serial femtosecond crystallography (SFX) with X-ray free electron lasers and microcrystal electron diffraction (MicroED) using electron cryomicroscopes. Once considered useless for structure determination, nano/microcrystals now confer significant advantages for static and time-resolved structure determination from a wide variety of difficult-to-study targets. MicroED has been used to obtain sub-Ångstrom resolution maps in which hydrogen atoms can be clearly resolved from only a few nano/microcrystals, while SFX has been used to probe protein dynamics following reaction initiation on time scales from femtoseconds to minutes. We review these two complementary techniques and their abilities for high-resolution structure determination.
Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl
into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ...ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.
Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic ...cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.
Rational structure-based drug design (SBDD) relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for ...lead-compound optimization via an iterative process. While SBDD has proven successful for many drug-discovery projects, its application to G protein-coupled receptors (GPCRs) has been limited owing to extreme difficulties with their crystallization. Here, a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands, taking advantage of the serial femtosecond crystallography approach, which obviates the need for large crystals and requires only submilligram quantities of purified protein. The method was applied to the human β
-adrenergic receptor, resulting in eight room-temperature co-crystal structures with six different ligands, including previously unreported structures with carvedilol and propranolol. The generality of the proposed method was tested with three other receptors. This approach has the potential to enable SBDD for GPCRs and other difficult-to-crystallize membrane proteins.
Time-resolved studies of biomacromolecular crystals have been limited to systems involving only minute conformational changes within the same lattice. Ligand-induced changes greater than several ...angstroms, however, are likely to result in solid-solid phase transitions, which require a detailed understanding of the mechanistic interplay between conformational and lattice transitions. Here we report the synchronous behavior of the adenine riboswitch aptamer RNA in crystal during ligand-triggered isothermal phase transitions. Direct visualization using polarized video microscopy and atomic force microscopy shows that the RNA molecules undergo cooperative rearrangements that maintain lattice order, whose cell parameters change distinctly as a function of time. The bulk lattice order throughout the transition is further supported by time-resolved diffraction data from crystals using an X-ray free electron laser. The synchronous molecular rearrangements in crystal provide the physical basis for studying large conformational changes using time-resolved crystallography and micro/nanocrystals.
Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity ...induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.
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