Published: June 5th, 2021
Here, we describe how to use the automated macromolecular crystallography pipelines for protein-to-structure, rapid ligand-protein complex analysis and large-scale fragment screening based on the CrystalDirect technology at the HTX Laboratory in EMBL Grenoble.
EMBL Grenoble operates the High Throughput Crystallization Laboratory (HTX Lab), a large-scale user facility offering high throughput crystallography services to users worldwide. The HTX lab has a strong focus in the development of new methods in macromolecular crystallography. Through the combination of a high throughput crystallization platform, the CrystalDirect technology for fully automated crystal mounting and cryocooling and the CRIMS software we have developed fully automated pipelines for macromolecular crystallography that can be remotely operated over the internet. These include a protein-to-structure pipeline for the determination of new structures, a pipeline for the rapid characterization of protein-ligand complexes in support of medicinal chemistry, and a large-scale, automated fragment screening pipeline enabling evaluation of libraries of over 1000 fragments. Here we describe how to access and use these resources.
Automation has been introduced at all steps of the macromolecular crystallography experimental process, from crystallization to diffraction data collection and processing1,2,3,4,5,6,7,8,9, including a number of technologies for sample mounting10,11,12,13,14,15,16,17. This has not only accelerated the pace at which crystallographic structures are obtained but has contributed to streamline applications like structure guided drug design18,19,20,21,22,23,24. In this manuscript we describe some of the aspects of the automated crystallography pipelines available at the HTX lab in Grenoble as well as the underlying technologies.
The HTX lab at EMBL Grenoble is one of the largest academic facilities for crystallization screening in Europe. It is co-located at the European Photon and Neutron (EPN) campus togetherwith the European Synchrotron Radiation Facility (ESRF), which produces some of the world's most brilliant X-ray beams and the Institut Laue Langevin (ILL), which provides high flux neutron beams. Since the start of operations in 2003 the HTX lab has provided services to over 800 scientists and processes more than 1000 samples per year. The HTX lab has a strong focus in the development of new methods in macromolecular crystallography, including methods for sample evaluation and quality control25,26 and the CrystalDirect technology, enabling fully automated crystal mounting and processing15,16,17. The HTX lab has also developed the Crystallographic Information Management System (CRIMS), a web-based laboratory information system that provides automated communication between crystallization and synchrotron data collection facilities, enabling uninterrupted information flow over the whole sample cycle from pure protein to diffraction data. Through the combination of the capacities of the HTX facility, the CrystalDirect technology and the CRIMS software, we have developed fully automated protein-to-structure pipelines integrating crystallization screening, crystal optimization, automated crystal harvesting processing and cryocooling and X-ray data collection at multiple synchrotrons into a single and continuous workflow that can be remotely operated through a web browser. These pipelines can be applied to support rapid determination of new structures, the characterization of protein-ligand complexes and large-scale compound and fragment screening through X-ray crystallography.
The HTX lab is equipped with a nonvolume crystallization robot (including an LCP module that enables crystallization of both soluble and membrane proteins), crystal farms (at 5 °C and 20 °C), two robotic liquid handling stations to prepare crystallization screens, and two automated CrystalDirect crystal harvesters with capacity to produce and store up to 400 frozen sample pins per operation cycle. Scientists send their samples to the facility by express courier, which are then processed by dedicated technicians at the HTX lab. Scientists can remotely design crystallization screening and optimization experiments through a web interface provided by the CRIMS system. Through this interface, they can choose from a wide range of parameters and experimental protocols available at the facility to fit their specific sample requirements. Results together with all experimental parameters are made available to users in real time through CRIMS. All samples received are assayed through a specifically developed method that enables to estimate the crystallization likelihood of the sample25,26,27. Based on the results of this assay specific recommendations are made to users concerning the optimal incubation temperature and possible sample optimization experiments. Once crystallization experiments are set up, scientist can evaluate the results by looking at crystallization images collected at different time points through the web. When crystals suitable for X-ray diffraction experiments are identified, scientists can use a dedicated interface to establish a crystal mounting plan that will then be executed by the CrystalDirect robot.
The CrystalDirect technology is based on the use of a modified vapor diffusion crystallization microplate and a laser beam to mount and cryo-cool crystal samples into diffraction compatible supports closing the automation gap existing between crystallization and data collection15,16,17. Briefly, crystals are grown in a modified vapour diffusion plate, the CrystalDirect microplate. Once crystals appear the CrystalDirect harvesting robot automatically applies a laser beam to excise a film piece containing the crystal, attach it to a standard diffraction data collection pin, and cryo-cool it in a nitrogen gas stream (see Zander et al. 2016 and https://www.youtube.com/watch?v=Nk2jQ5s7Xx8 ). This technology has a number of additional advantages over manual or semi-automated crystal mounting protocols. For example, the size and shape of the crystals is not an issue, making it equally easy to harvest large crystals or microcrystals, it is often possible to avoid the use of cryo-protectants, due to the special way in which the technology operates (see reference 17, Zander et al.), making X -ray diffraction analysis much more straightforward. The laser-beam can also be used as a surgical tool to select the best parts of a sample when crystals grow on clusters or show epitaxial growth for example. The CrystalDirect technology can also be used to automated soaking experiments17. Delivery of solutions with small molecules or other the chemicals to crystals. Thereby it enables to support fully automated, large-scale compound and fragment screening. Once Crystals are harvested and cryocooled by the CrystalDirect robot, they are transferred to either SPINE or Unipuck pucks which are compatible with most synchrony macromolecular crystallography beamlines around the world. The system can harvest up to 400 pins (the capacity of the cryogenic storage Dewar) in a fully autonomous manner. CRIMS communicates with the harvester robot during the process and provides automated tracking of crystal samples (pucks and pins). Pucks are marked with both barcodes and RFID tags to facilitate sample management21,28.
CRIMS provides an application program interface (API) supporting automated communication with the ISPyB system supporting X-ray data collection management and processing at many synchrotrons in Europe and the world29. After automated crystal harvesting is completed, scientists can select crystal samples (pucks) and create sample shipments for the macromolecular crystallography beamlines at either the ESRF (Grenoble, France)7,8,9 or Petra III synchrotrons (Hamburg, Germany)18,19. CRIMS transfer the data corresponding to the selected beamline samples to the synchrotron information system along with pre-selected data collection parameters. Once the samples arrive at the selected synchrotron beamline, X-ray data collection is carried out either manually, through remote beamline operation or in a fully automated manner (i.e., at the MASSIF-1 beamline of the ESRF8 operated by the joint EMBL ESRF Joint Structural Biology Group (JSBG)). After data collection CRIMS retrieves automatically information about the results of data collection along with initial data processing results carried out by the synchrotron data processing systems and presents it to the scientist through a convenient user interface.
The HTX lab applies these automated pipelines to support three different applications, rapid determinations of new structures, rapid characterization of protein-ligand complexes and large-scale compound and fragment screening. Below we describe the how to use and operate them.
NOTE: Funded access to these pipelines for scientists worldwide is supported through a series of funding programs. At the moment of writing this manuscript applications for access are accepted through either the iNEXT Discovery program (https://inext-discovery.eu), an European facility network to stimulate translational structural biology20 funded by the Horizon 2020 programme of the European Commission or INSTRUCT-Eric (https://instruct-eric.eu/). Contact the corresponding author for the current modalities and routes for funded access at a particular time. This protocol describes operation of the protein-to-structure pipeline and includes steps common to all our pipelines while specificities for the other two pipelines are discussed in the following section. The instructions here refer to CRIMS V4.0.
1. High Throughput Crystallization Laboratory
The automated crystallography pipeline described above has been applied to support a big number of internal and external projects with remarkable success. A few highlights include the project from Djinović-Carugo and co-workers from the Max Perutz Laboratories (Vienna) focusing on the structural and functional analysis of a dipeptidyl peptidase essential for the growth a bacterial pathogen. The rapid succession of crystallization screening, diffraction evaluation, crystal optimization and X-ray data collection cycles (up to 8 iterations for this project) enabled to obtain structural models for three different conformational states of the protein in just a few weeks, which provided key mechanistic understanding on the function of this class of proteins36 (see Figure 1).
Another example is the from Macias and co-workers from the Institute of Biomedical Research (IRB, Barcelona) that combined bioinformatics tools and structural approaches to identify new DNA binding motifs for the SMAD3 and SMAD4 transcription factors involved in cell fate regulation. This work has produced 6 high resolution structures of SMAD3 & 4 in complex with different DNA binding motifs37,38 revealing a so far unsuspected capacity of these transcription factors to recognize and bind to a diverse array of DNA sequences, which is key for the interpretation of their function in different biological contexts. These technologies have also been applied to support proprietary research in the context of drug design projects from research groups in pharma and biotech companies. For example, thanks to the rapidity contributed by these pipelines, the structural analysis of multiple ligand-target complexes can be achieved within days, which is of great value to support successive rounds medicinal chemistry optimization in the context of drug development. Finally we have also applied this infrastructure for large-scale X-ray based fragment screening39.
Figure 1: Automated Crystallography Pipeline. Integrated operation of the EMBL HTX lab including the CrystalDirect technology and the CRIMS software with the MASSIF-1 beamline at ESRF and automated communication between the CRIMS and ISPyB software enable to support fully automated, remote controlled protein-to-structure pipeline integrating crystallization screening and optimization, automated crystal harvesting and cryo-cooling and automated data collection and processing. The structural models correspond to three different conformational states of a protease from a pathogenic bacterium identified in a record time by applying these pipeline36. Please click here to view a larger version of this figure.
The automated crystallography pipelines described here are available to researchers worldwide through different funding programs. Currently, funded access for crystallization experiments and the CrystalDirect technology can be obtained by applying to the iNEXT Discovery program and INSTRUCT-ERIC, while access to macromolecular crystallography beamlines at the ESRF is supported through the ESRF user access program. This approach minimizes the delay between crystal growth and measurement, accelerating the progression of very challenging projects that require diffraction-based optimization of protein production and crystallization conditions and frees scientists from complex operations associated with crystallization, crystal handling and beamline operation, rendering crystallography more accessible to non-expert groups. It can also be used for rapid exploration of crystallization additives, phasing agents or for compound screening through co-crystallization experiments. While most crystallography projects could potentially benefit from this approach, some samples may require special protocols not amenable to automation or to the pipelines presented here, for example those requiring microfluidic systems or highly specialized crystallization devices or samples that are extremely labile and would not tolerate shipment.
The CrystalDirect technology also enables automated crystal soaking17 for the characterization of of small molecule-target complexes. For this, a small aperture is created with the laser prior to the harvesting process and a drop of a solution containing the desired chemicals (i.e., phasing agents or potential ligands) is added on top, so that it enters in contact with, and diffuses into the crystallization solution eventually reaching the crystal. Chemical solutions can be formulated in water, DMSO or other organic solvents. After a certain incubation time the crystals can be harvested and analyzed by diffraction as described above. This approach has been applied to the rapid characterization of ligand-protein complexes in the context of structure-based drug design as well as to large-scale compound and fragment screening. In the latter case fragment libraries with hundreds to over a thousand fragments can be rapidly analyzed. Specific CRIMS interfaces not presented here facilitate the design and automated tracking of crystal soaking experiments, while integration between the CRIMS software and the Pipedream software suite, developed by Global Phasing Ltd (U.K) enable automated data processing, phasing, ligand identification and structure refinement over hundreds of datasets in parallel, streamlining data analysis and interpretation32,33. For example, this pipeline was recently applied to the identification of fragments binding both to the active site and several allosteric sites of Trypanosoma brucei farnesyl pyrophosphate synthase, a key enzyme of the parasite causing human African trypanosomiasis.
The pipelines presented here can contribute to accelerate the pace of discovery in structural biology and make macromolecular crystallography more accessible to a larger number of research groups. Moreover, by facilitating large-scale compound and fragment screening they can contribute to foster translational research and speed up the process of drug discovery, contributing to facilitate the development of better and safer drugs against a larger number of targets.
JAM is co-author of a patent on the CrystalDirect system
We want to thank the joint EMBL-ESRF Structural Biology Group (JSBG) for support in the use and operation of the ESRF macromolecular beamlines. We are thankful to Matthew Bowler for support with data collection at the MASSIF-1 beamline of ESRF and Thomas Schneider and the EMBL Hamburg Team for excellent support with data collection at P14 of the PetraIII synchrotron (DESY, Hamburg, Germany). The CrystalDirect harvester is developed in collaboration with the Instrumentation Team at EMBL Grenoble. This project was supported by funding from the European CommunityH2020 Programme under the projects iNEXT (Grant No 653706) and iNEXT Discovery (Grant No 871037) as well as the Région Auvergne-Rhône-Alpes through the Booster programme.
|Automated crystal mounting and cryocooling
|CrystalDirect Crystallization plate
|96-well crytsallization microplate
|For the autoamted preparation of crystallization screens
|Mosquito crystallization Robot
|For the preparation of crystallization experiments
|Tecan Evo Liquid handling station
|For the preparation of crystallization solutions
|SPINE-compatible cryogenic pucks for automated synchrotron sample exchangers
|Universal cryogenic pucks for automated synchrotron sample exchangers
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