An electropneumatic cleaning device for piezo-actuator-driven picolitre-droplet dispensers.

Recently, we introduced the liquid application method for time-resolved analyses (LAMA). The time-consuming cleaning cycles required for the substrate solution exchange and storage of the sensitive droplet-dispenser nozzles present practical challenges. In this work, a dispenser cleaning system for the semi-automated cleaning of the piezo-actuator-driven picolitre-droplet dispensers required for LAMA is introduced to streamline typical workflows.

Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography.

We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with a time-resolution in the millisecond range. Protein crystals are mounted on canonical micromeshes on an electropneumatic piston, where the crystals are kept in a humidity and temperature-controlled environment, then reactions are initiated via the liquid application method (LAMA) and plunging into liquid nitrogen is initiated after an electronically set delay time to cryo-trap intermediate states. High-magnification images are automatically recorded before and after droplet deposition, prior to plunging. The SPINE-standard sample holder is directly plunged into a storage puck, enabling compatibility with high-throughput infrastructure. Here we demonstrate binding of glucose and 2,3-butanediol in microcrystals of xylose isomerase, and of avibactam and ampicillin in microcrystals of the extended spectrum beta-lactamase CTX-M-14. We also trap reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase to demonstrate that the spitrobot enables insight into catalytic events.

Environmental Liquid Cell Technique for Improved Electron Microscopic Imaging of Soft Matter in Solution.

Liquid-phase transmission electron microscopy is a technique for simultaneous imaging of the structure and dynamics of specimens in a liquid environment. The conventional sample geometry consists of a liquid layer tightly sandwiched between two Si3N4 windows with a nominal spacing on the order of 0.5 µm. We describe a variation of the conventional approach, wherein the Si3N4 windows are separated by a 10-µm-thick spacer, thus providing room for gas flow inside the liquid specimen enclosure. Adjusting the pressure and flow speed of humid air inside this environmental liquid cell (ELC) creates a stable liquid layer of controllable thickness on the bottom window, thus facilitating high-resolution observations of low mass-thickness contrast objects at low electron doses. We demonstrate controllable liquid thicknesses in the range 160 ± 34 to 340 ± 71 nm resulting in corresponding edge resolutions of 0.8 ± 0.06 to 1.7 ± 0.8 nm as measured for immersed gold nanoparticles. Liquid layer thickness 40 ± 8 nm allowed imaging of low-contrast polystyrene particles. Hydration effects in the ELC have been studied using poly-N-isopropylacrylamide nanogels with a silica core. Therefore, ELC can be a suitable tool for in situ investigations of liquid specimens.

The HARE chip for efficient time-resolved serial synchrotron crystallography.

Serial synchrotron crystallography (SSX) is an emerging technique for static and time-resolved protein structure determination. Using specifically patterned silicon chips for sample delivery, the `hit-and-return' (HARE) protocol allows for efficient time-resolved data collection. The specific pattern of the crystal wells in the HARE chip provides direct access to many discrete time points. HARE chips allow for optical excitation as well as on-chip mixing for reaction initiation, making a large number of protein systems amenable to time-resolved studies. Loading of protein microcrystals onto the HARE chip is streamlined by a novel vacuum loading platform that allows fine-tuning of suction strength while maintaining a humid environment to prevent crystal dehydration. To enable the widespread use of time-resolved serial synchrotron crystallography (TR-SSX), detailed technical descriptions of a set of accessories that facilitate TR-SSX workflows are provided.

Liquid application method for time-resolved analyses by serial synchrotron crystallography.

We introduce a liquid application method for time-resolved analyses (LAMA), an in situ mixing approach for serial crystallography. Picoliter-sized droplets are shot onto chip-mounted protein crystals, achieving near-full ligand occupancy within theoretical diffusion times. We demonstrate proof-of-principle binding of GlcNac to lysozyme, and resolve glucose binding and subsequent ring opening in a time-resolved study of xylose isomerase.