The Automated Crystallography Pipelines at the EMBL HTX Facility in Grenoble

The HTX lab offers online access to fully automated protein to structured pipelines that can be applied to solve new structures, and to carry out larger scale fragment and legan screening. Through the combination of the CrystalDirect technology for automated crystal harvesting and CRI cooling on the CRIM software, where scientists can control the whole crystallography process over the internet. Begin by opening and logging into the Crystal Graphic Information Management System in a web browser and click the Samples Menu to open an interface with the project in Sample Management Tools.

Click new sample and in the sample window, enter the requested information and click save and make request. In the Request Panel, select the crystallization protocol, the incubation temperature, the crystallization screens to be used, and the desired date for the experiments that should be compatible with the time required for the sample shipment. Use the comment fields to provide indications about the samples that are important for the HDX lab operators to know.

After submission, the crystallization request will be validated by the High Throughput Crystallization Lab team. A confirmation about scheduling the experiment will be sent via email. Upon the arrival of the sample to the facility.

The lab personnel will set up the experiment, send confirmation of the setup via email, and transfer the crystallization trays to the automated imagers. CRIMS will provide access to all the experimental parameters and track new imaging sessions in real time. When new images are available, email notifications will be sent automatically.

To see the images from the Crystallization Plates, open the Plates Menu and navigate to the sample. Click view to see the last imaging session or click the expand symbol to select a different imaging session and check the drop history. To filter samples for a selected project.

Click on the specific project box. Search functions are also available for most of the table columns. To evaluate and score the results of the crystallization experiments, select the plate view interface, and navigate through the different walls of the crystallization plates.

Select the image type and select the image quality or record the scores. The view interface also provides all of the experimental parameters used for the crystallization experiments, including the composition of the crystallization solution. To design Crystal Optimization Screens based on the primary hit conditions identified through the initial screening, open the Refinement Menu and select Screens sub menu.

Select the plate type, stock solutions or gradient configurations that best fit the experimental design. It is possible to ask CRIMS to output a file directly compatible with the formulator robot formulatrix to automatically pipette the screens into the plate or to output a printable document but the volumes for manual operation. Once crystal is suitable for X-ray diffraction experiments have been identified, navigate to the Plate View Interface and use the pre stored scores to select the image corresponding to the correct crystallization drop.

Click crystal harvesting to record an automated crystal harvesting plan from the crystal direct harvester robot. CRIMS will automatically record and store the location of the harvested crystals into either spine or unit pucks. Next, operators that our facility will remove the plates with the harvesting plans from the imagers and transfer them to the CrystalDirect robot.

After crystal harvesting is completed, the pucks containing the samples are placed into a CX 100 Dewar or transferred to the Synchrotron of your choice. To inspect the cryo cooled samples, open the Crystal Manager Menu and select Harvested Crystals. The images of the harvesting process, including the images of the pins with the harvested crystals will be available there.

To create sample shipments for X-ray diffraction analysis, open the Shipments Menu and click create shipment to select the Synchrotron of interest and the bag number. Comments to support the data collection as well as predefined parameters for data collection at fully-automated beamlines can also be entered. The HDX lab also provides access to fully automated large-scale fragments screening.

CRIMS resources for automated data processing and hit identification based on the global phasing software, Pipeline Pipe Dream, make it possible to rapidly evaluate and prioritize results over hundreds to thousands of datasets in a single project. CRIMS communicates with the Synchrotron information management systems and retrieves information about the actual diffraction experiments. Open the Crystal Manager Menu and select crystal diffraction data to view summary information and the results of the initial data processing at the Synchrotron.

In this representative analysis, the automated crystallography pipelines were used as demonstrated, for the structural identification of different proteins. As observed, three different confirmational states of a protease from a pathogenic bacterium were identified in a very short time, thanks to the rapid succession of crystal screening optimization and diffraction measurements. This pipeline was also recently applied to the identification of fragments, binding both to the active in several alasteric sites of Trypanosoma brucei.

Farnesyl pyrophosphate synthase a key enzyme of the parasite causing human African trypanosomiasis. This process minimizes the delay between crystal growth and measurement, accelerating progress. It also frees scientists from complex operations like crystal handling and beamline operation rendering crystallography, more accessible to non-experts.

This approach can contribute to foster translational research and then speed up the process of drug discovery. Contributing to the development of better and safer drugs against a larger number of targets.