Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme.

Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.

3D printing of gas-dynamic virtual nozzles and optical characterization of high-speed microjets.

Gas dynamic virtual nozzles (GDVNs) produce microscopic flow-focused liquid jets and droplets and play an important role at X-ray free-electron laser (XFEL) facilities where they are used to steer a stream of hydrated biomolecules into an X-ray focus during diffraction measurements. Highly stable and reproducible microjet and microdroplets are desired, as are flexible fabrication methods that enable integrated mixing microfluidics, droplet triggering mechanisms, laser illumination, and other customized features. In this study, we develop the use of high-resolution 3D nano-printing for the production of monolithic, asymmetric GDVN designs that are difficult to fabricate by other means. We also develop a dual-pulsed nanosecond image acquisition and analysis platform for the characterization of GDVN performance, including jet speed, length, diameter, and directionality, among others. We show that printed GDVNs can form microjets with very high degree of reproducibility, down to sub-micron diameters, and with water jet speeds beyond 170 m/s.

Three-dimensional-printed gas dynamic virtual nozzles for x-ray laser sample delivery.

Reliable sample delivery is essential to biological imaging using X-ray Free Electron Lasers (XFELs). Continuous injection using the Gas Dynamic Virtual Nozzle (GDVN) has proven valuable, particularly for time-resolved studies. However, many important aspects of GDVN functionality have yet to be thoroughly understood and/or refined due to fabrication limitations. We report the application of 2-photon polymerization as a form of high-resolution 3D printing to fabricate high-fidelity GDVNs with submicron resolution. This technique allows rapid prototyping of a wide range of different types of nozzles from standard CAD drawings and optimization of crucial dimensions for optimal performance. Three nozzles were tested with pure water to determine general nozzle performance and reproducibility, with nearly reproducible off-axis jetting being the result. X-ray tomography and index matching were successfully used to evaluate the interior nozzle structures and identify the cause of off-axis jetting. Subsequent refinements to fabrication resulted in straight jetting. A performance test of printed nozzles at an XFEL provided high quality femtosecond diffraction patterns.

Room-temperature macromolecular serial crystallography using synchrotron radiation.

A new approach for collecting data from many hundreds of thousands of microcrystals using X-ray pulses from a free-electron laser has recently been developed. Referred to as serial crystallography, diffraction patterns are recorded at a constant rate as a suspension of protein crystals flows across the path of an X-ray beam. Events that by chance contain single-crystal diffraction patterns are retained, then indexed and merged to form a three-dimensional set of reflection intensities for structure determination. This approach relies upon several innovations: an intense X-ray beam; a fast detector system; a means to rapidly flow a suspension of crystals across the X-ray beam; and the computational infrastructure to process the large volume of data. Originally conceived for radiation-damage-free measurements with ultrafast X-ray pulses, the same methods can be employed with synchrotron radiation. As in powder diffraction, the averaging of thousands of observations per Bragg peak may improve the ratio of signal to noise of low-dose exposures. Here, it is shown that this paradigm can be implemented for room-temperature data collection using synchrotron radiation and exposure times of less than 3 ms. Using lysozyme microcrystals as a model system, over 40 000 single-crystal diffraction patterns were obtained and merged to produce a structural model that could be refined to 2.1 Šresolution. The resulting electron density is in excellent agreement with that obtained using standard X-ray data collection techniques. With further improvements the method is well suited for even shorter exposures at future and upgraded synchrotron radiation facilities that may deliver beams with 1000 times higher brightness than they currently produce.

Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser.

The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.

Single mimivirus particles intercepted and imaged with an X-ray laser.

X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.

Radiation damage in protein serial femtosecond crystallography using an x-ray free-electron laser.

X-ray free-electron lasers deliver intense femtosecond pulses that promise to yield high resolution diffraction data of nanocrystals before the destruction of the sample by radiation damage. Diffraction intensities of lysozyme nanocrystals collected at the Linac Coherent Light Source using 2 keV photons were used for structure determination by molecular replacement and analyzed for radiation damage as a function of pulse length and fluence. Signatures of radiation damage are observed for pulses as short as 70 fs. Parametric scaling used in conventional crystallography does not account for the observed effects.