Double-flow focused liquid injector for efficient serial femtosecond crystallography

Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow- focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices

Femtosecond x-ray diffraction from an aerosolized beam of protein nanocrystals

We demonstrate near-atomic-resolution Bragg diffraction from aerosolized single granulovirus crystals using an x-ray free-electron laser. The form of the aerosol injector is nearly identical to conventional liquid-microjet nozzles, but the x-ray-scattering background is reduced by several orders of magnitude by the use of helium carrier gas rather than liquid. This approach provides a route to study the weak diffuse or lattice-transform signal arising from small crystals. The high speed of the particles is particularly well suited to upcoming MHz-repetition-rate x-ray free-electron lasers

Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus 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 binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

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^(pro)), 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^(pro). In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2

Experimental and Numerical Investigation of Gas-Focused Liquid Micro-Jet Velocity

Compressible multiphase numerical simulations of gas-focused micro-jets are compared with the experimental data obtained with the dual pulse imaging laser-induced fluorescence drop velocimetry. Such jets, originating from a 3D printed gas dynamic virtual nozzle into a low-vacuum (150 Pa) environment, are increasingly being used for sample delivery in serial femtosecond crystallography. The distance traveled by a detaching drop from the jet is measured between the two consecutive illumination pulses with a known time delay at the positions 200 µm and 450 µm from the nozzle. Additionally, the high-speed camera images are used to analyze the shape of the jet. An axisymmetric, compressible, Newtonian two-phase helium-water mixture model is numerically solved within the framework of the volume of fluid and the finite volume method. The experimental and the computational studies are performed with a constant volumetric liquid flow rate of 14 μl/min and the gas mass flow rate in the range from 4.6 mg/min to 20 mg/min. The related jet Reynolds number ranges from 120 to 220 and Weber number from 30 to 150. The maximum difference between the measurements and the results of the numerical model in terms of the droplet velocity and jet diameter is within 10 %. The study provides new information on the jet velocities for micron-sized gas-focused nozzles. The validated numerical model can be used as a design tool for the nozzles dedicated to the specific needs of the femtosecond crystallography experiments

Mix-and-Inject Serial Femtosecond Crystallography to Capture RNA Riboswitch Intermediates

Time-resolved structure determination of macromolecular conformations and ligand-bound intermediates is extremely challenging, particularly for RNA. With rapid technological advances in both microfluidic liquid injection and X-ray free electron lasers (XFEL), a new frontier has emerged in time-resolved crystallography whereby crystals can be mixed with ligand and then probed with X-rays (mix-and-inject) in real time and at room temperature. This chapter outlines the basic setup and procedures for mix-and-inject experiments for recording time-resolved crystallographic data of riboswitch RNA reaction states using serial femtosecond crystallography (SFX) and an XFEL

Validation of a Computational Model for a Coupled Liquid and Gas Flow in Micro-Nozzles

The work presents verification of a numerical model for micro-jet focusing, where a coupled liquid and gas flow occurs in a gas dynamic virtual nozzle (GDVN). Nozzlesof this type are usedinserial femtosecond crystallographyexperimentsto deliver samplesintoX-ray beam. Thefollowing performance criteria are desirable: the jet to be longer than 100 μm to avoid nozzle shadowing, the diameter as small as possibleto minimize the background signal,and the jet velocityas high as possible to avoid sample’sdouble X-ray exposure.Previouscomprehensive numerical investigation has been extended to includenumerical analysis of the tip jet velocities. These simulations were then comparedwith the experimental data. The coupled numerical model of a 3D printed GDVN considers a laminar two-phase, Newtonian, compressible flow, which is solved based on the finite volume method discretization and interface tracking with volume of fluid (VOF). The numerical solution is calculated with OpenFOAM based compressible interFoam solver, which uses algebraicformulation of VOF. In experimental setupfor model validationa 3D printed GDVN was inserted in a vacuum chamber with two windows used for illumination and visualization. Once the jet was stabilized its velocity wasestimated based on a distance a droplet traveled between two consecutive illumination pulseswith a known time delay. The experimental and computational study was performed for a constant liquid flow rate of 14 $\mu$l/min and the gas mass flow rate in the range from 5 mg/min to 25 mg/min. The coupled numerical model reasonablypredictsthe jet speed and shape as a function of the gas flow

The Natural Breakup Length of a Steady Capillary Jet: Application to Serial Femtosecond Crystallography

One of the most successful ways to introduce samples in Serial Femtosecond Crystallography has been the use of microscopic capillary liquid jets produced by gas flow focusing, whose length-to-diameter ratio and velocity are essential to fulfill the requirements of the high pulse rates of current XFELs. In this work, we demonstrate the validity of a classical scaling law with two universal constants to calculate that length as a function of the liquid properties and operating conditions. These constants are determined by fitting the scaling law to a large set of experimental and numerical measurements, including previously published data. Both the experimental and numerical jet lengths conform remarkably well to the proposed scaling law. We show that, while a capillary jet is a globally unstable system to linear perturbations above a critical length, its actual and shorter long-term average intact length is determined by the nonlinear perturbations coming from the jet breakup itself. Therefore, this length is determined solely by the properties of the liquid, the average velocity of the liquid and the flow rate expelled. This confirms the very early observations from Smith and Moss 1917, Proc R Soc Lond A Math Phys Eng, 93, 373, to McCarthy and Molloy 1974, Chem Eng J, 7, 1, among others, while it contrasts with the classical conception of temporal stability that attributes the natural breakup length to the jet birth conditions in the ejector or small interactions with the environment

The natural breakup length of a steady capillary jet

72 American Physical Society Division of Fluid Dynamics 2019Despite their fundamental and applied importance, a general model to predict the natural breakup length of steady capillary jets has not been proposed yet. In this work, we derive a scaling law with two universal constants to calculate that length as a function of the liquid properties and operating conditions. These constants are determined by fitting the scaling law to a large set of experimental and numerical measurements, including previously published data. Both the experimental and numerical jet lengths conform remarkably well to the proposed scaling law. This law is explained in terms of the growth of perturbations excited by the jet breakup itself

Post-sample aperture for low background diffraction experiments at X-ray free-electron lasers

The success of diffraction experiments from weakly scattering samples strongly depends on achieving an optimal signal-to-noise ratio. This is particularly important in single-particle imaging experiments where diffraction signals are typically very weak and the experiments are often accompanied by significant background scattering. A simple way to tremendously reduce backgroundscattering by placing an aperture downstream of the sample has been developed and its application in a single-particle X-ray imaging experiment at FLASH is demonstrated. Using the concept of a post-sample aperture it was possible to reduce the background scattering levels by two orders of magnitude