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 R&#233;gion Auvergne-Rh&#244;ne-Alpes through the Booster programme.

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&#160;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.&#160;
1. High Throughput Crystallization Laboratory
<ol>
	<li>Before starting, ask for registration at the HTX lab through the CRIMS system https://htxlab.embl.fr/#/. The user credentials provide remote access to all experimental design and evaluation interfaces.&#160;
	<ol>
		<li>Log in to CRIMS through a web navigator (Firefox, Chrome and Safari are supported). The CRIMs web server is encrypted to prevent third parties from accessing data while it travels through the web. Once in CRIMS, a series of menus to the left of the screening help manage and create samples, request crystallization experiments, manage and visualize plates, etc. A series of video tutorials are available at https://medias01-web.embl.de/Mediasite/Showcase/embl/Channel/a2168bcaa36b4564851663e5b69594014d. 
		NOTE: Users sending samples by courier need to register the samples and the requested crystallization experiments through CRIMS before sending the sample to ensure they can be processed without delay upon arrival. Please, send the shipment details to htx@embl.fr.</li>
	</ol>
	</li>
	<li>Log in into CRIMS (https://htxlab.embl.fr ) with a web browser and click on the&#160;Samples Menu. This will open an interface with project and sample management tools.</li>
	<li>Click on the New Sample button and provide the information requested. CRIMS allows to organize samples under different projects, targets and constructs. Assign the sample to existing ones or create new ones at this point.</li>
	<li>Once the requested information has been entered click on Save &#38; Make Request. Select the crystallization protocol, the crystallization screens to be used, the incubation temperature and the desired date for the experiments.
	<ol>
		<li>Use the comment fields to provide indications about the samples that are important for the HTX lab operators to know. Custom screens can also be selected (see below). After submitting the crystallization request it will be validated by the HTX Lab Team and confirmation about the scheduling of the experiments will be sent via e-mail. Make sure to select experiment dates that are compatible with times required for sample shipment.</li>
	</ol>
	</li>
	<li>Once the samples arrive in the facility operators at the HTX lab will carry out the experiments as requested. Once crystallization experiments are set up, confirmation will be sent via e-mail and crystallization trays will be transferred to the automated imagers. CRIMS provides access to all experimental parameters and will automatically track new imaging sessions. E-mail notifications will be automatically sent when new images are available. An thermofluor-based30&#160;sample quality assessment experiment based on a protocol developed at the facility25,26&#160;is carried out with every sample at this point and will be available through CRIMS.</li>
	<li>Images of the crystallization experiments along with the results of the sample quality assessment will be available in CRIMS shortly after the crystallization trays are set up. Click on the Thermofluor menu and navigate to the sample to see the results of the sample quality assessment experiment.
	<ol>
		<li>Click on the Plates menu to see the images from the Crystallization plates. Navigate to the sample and either click on View to see the last imaging session or on the + (expand) symbol to select a different imaging session. A series of tools help to find and navigate easily through the samples. For example, clicking in a project box at the top of the screens filters samples for that project and search functions are available for most of the table columns.</li>
	</ol>
	</li>
	<li>Use the plate View interface to help evaluate and score the results of the crystallization experiments. It allows navigation through the different wells of the crystallization plates, select image types (i.e., Vis, UV), select image resolution or record scores, for example. This interface also provides all experimental parameters used for the crystallization experiments including the composition of the crystallization solutions.</li>
	<li>Click on the Refinement menu to design crystal optimization screens based on primary hit conditions identified through the initial screening. The Chemicals and Stock Solutions submenus allow one to register and manage the crystallization stock solutions. The Screens submenu provides access to an interface to design your own optimization or custom screens.
	<ol>
		<li>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 with the volumes for manual operation.</li>
	</ol>
	</li>
	<li>Iterate through steps 1.2-1.8 to carry out crystal optimization experiments.</li>
	<li>Once crystals suitable for X-ray diffraction experiments are identified, navigate to the plate View interface and select the image corresponding to the right crystallization drop. Pre-stored scores will help you do this easily.
	<ol>
		<li>Click either on Crystal Harvesting to record an automated crystal harvesting plan for the CrystalDirect harvester robot or on Manual Harvesting for traditional manual crystal mounting, if using CRIMS at a facility that is not equipped with CrystalDirect. Both interfaces will guide the user through the crystal harvesting process. CRIMS will automatically record and store the location of harvested crystals into either SPINE or Unipucks21.</li>
	</ol>
	</li>
	<li>Select the Crystal Manager menu in CRIMS. Click on the Harvested Crystals submenu to inspect the frozen samples. When using the CrystalDirect harvester, images of the harvesting process are presented, including images of the pins with the harvested crystals.</li>
	<li>Select the Shipments menu to connect to either ESRF or Petra III synchrotrons and create sample shipments for X-ray diffraction analysis. Click on the Create Shipment button and select the synchrotron you want to use and the bag number (the bag password at the synchrotron is necessary here). The next series of interfaces are used to select the pukcs to be included in the shipment. The system makes it possible to provide comments to support data collection and determine data collection parameters for automated beamlines like MASSIF-1.</li>
	<li>If data collection is being carried out at ESRF or Petra III HTX lab, operators will transfer the samples to the beamline, data collection at other synchrotrons will be done at the user&#39;s own expense. It is possible to collect data by traveling to the synchrotron, through remote beamline operation or at MASSIF-1. In the latter case, the data collection process is fully automated. At the synchrotron, specific interfaces in ISPyB29&#160;allow users to recover the information sent by CRIMS and associate sample pucks to it so that results of data collection are automatically tracked. For the experiments described here, data collection at synchrotrons was typically carried out with the MXcube31&#160;software, while data processing and structure refinement was carried out with atuoPROC32, Staraninso33, BUSTER33, Pipedream32,33&#160;and Coot35.</li>
	<li>Once data collection experiments have been carried out CRIMS retrieves summary information along with results of initial data processing at the synchrotron from the ISPyB29&#160;system. Go to CRIMS Crystal Manager menu and click on the Crystal Diffraction Data sub menu. All the information and metadata regarding diffraction data collection is available. It is also possible to download processed data from the synchrotron as well as raw diffraction images. View multiple data collections or select specific datasets. Sample management tools make it possible to navigate and select samples for specific projects constructs. 
	NOTE: This pipeline provides fully automated operation over the internet from pure protein to X-ray diffraction results and can be operated with one or multiple samples at the same time. It can be applied to different context and project types in structural biology.</li>
</ol>

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JAM is co-author of a patent on the CrystalDirect system

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&#160;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.

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&#39;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&#160;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 &#176;C and 20 &#176;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&#160;). 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&#160;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.

<table><tbody><tr><td>CrystalDirect harvester</td><td>Arinax</td><td></td><td>Automated crystal mounting and cryocooling</td></tr><tr><td>CrystalDirect Crystallization plate</td><td>Mitegen</td><td>SKU: M-XDIR-96-2</td><td>96-well crytsallization microplate</td></tr><tr><td>Formulator 16</td><td>Formulatrix</td><td></td><td>For the autoamted preparation of crystallization screens</td></tr><tr><td>Mosquito crystallization Robot</td><td>SPT Labtech</td><td></td><td>For the preparation of crystallization experiments</td></tr><tr><td>Tecan Evo Liquid handling station</td><td>Tecan</td><td></td><td>For the preparation of crystallization solutions</td></tr><tr><td>Spine Pucks</td><td>Mitegen</td><td>SKU: M-SP-SC3-1</td><td>SPINE-compatible cryogenic pucks for automated synchrotron sample exchangers</td></tr><tr><td>UniPucks</td><td>Mitegen</td><td>SKU: M-CP-111-021</td><td>Universal cryogenic pucks for automated synchrotron sample exchangers</td></tr></tbody></table>

the automated crystallography pipelines at the embl htx facility in 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.

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 Djinovi&#263;-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&#160;(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 &#38; 4 in complex with different DNA binding motifs37,38&#160;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62491/62491fig01.jpg" /> 
Figure 1:&#160;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. <a href="https://www.jove.com/files/ftp_upload/62491/62491fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Automated Crystallography Pipelines at the EMBL HTX Facility in Grenoble at JoVE.com

The Automated Crystallography Pipelines at the EMBL HTX Facility in Grenoble

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 ...

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JoVE Journal

Biochemistry

3.5K Views.  European Molecular Biology Laboratory. Het HTX-lab biedt online toegang tot volledig geautomatiseerde eiwit- tot gestructureerde pijpleidingen die kunnen worden toegepast om nieuwe structuren op te lossen en om grootschalige fragment- en leganscreening uit te voeren. Door de combinatie van de CrystalDirect-technologie voor geautomatiseerde kristaloogst en CRI-koeling op de CRIM-software, waar wetenschappers het hele kristallografieproces via internet kunnen regelen. Begin met het openen van en inloggen op het Crystal Graphic Infor...

Video: The Automated Crystallography Pipelines at the EMBL HTX Facility in Grenoble

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological macromolecules. Until recently, a major requirement has been the availability of relatively large, well diffracting crystals, which are often challenging to obtain. However, the advent of serial crystallography and a renaissance in multi-crystal data collection methods has meant that the availability of large crystals need no longer be a limiting factor. Here, we illustrate the use of the automated MeshAndCollect protocol, which first identifies the positions of many small crystals mounted on the same sample holder and then directs the collection from the crystals of a series of partial diffraction data sets for subsequent merging and use in structure determination. MeshAndCollect can be applied to any type of micro-crystals, even if weakly diffracting. As an example, we present here the use of the technique to solve the crystal structure of the Cyan Fluorescent Protein (CFP) Cerulean.

We present the use of the MeshAndCollect protocol to obtain a complete diffraction data set, for use in subsequent structure determination, composed of partial diffraction data sets collected from many small crystals of the fluorescent protein Cerulean.

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Chemistry

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results.
Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY&#160; II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input.
Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.

Crystallographic fragment screening at the Helmholtz-Zentrum Berlin is performed using a workflow with dedicated compound libraries, crystal handling tools, fast data collection facilities&#160;and largely automated data analysis. The presented protocol intends to maximize the output of such experiments to provide promising starting points for downstream structure-based ligand design.

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are ...

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise.
Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. 
We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines.

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source

In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify chemical entities that can be developed into potent drug candidates. Since the compounds are small, interactions are weak, and the screening method must therefore be highly sensitive; moreover, structural information tends to be crucial for elaborating these hits into lead-like compounds. Therefore, protein crystallography has always been a gold-standard technique, yet historically too challenging to find widespread use as a primary screen.
Initial XChem experiments were demonstrated in 2014 and then trialed with academic and industrial collaborators to validate the process. Since then, a large research effort and significant beamtime have streamlined sample preparation, developed a fragment library with rapid follow-up possibilities, automated and improved the capability of I04-1 beamline for unattended data collection, and implemented new tools for data management, analysis and hit identification.
XChem is now a facility for large-scale crystallographic fragment screening, supporting the entire crystals-to-deposition process, and accessible to academic and industrial users worldwide. The peer-reviewed academic user program has been actively developed since 2016, to accommodate projects from as broad a scientific scope as possible, including well-validated as well as exploratory projects. Academic access is allocated through biannual calls for peer-reviewed proposals, and proprietary work is arranged by Diamond's Industrial Liaison group. This workflow has already been routinely applied to over a hundred targets from diverse therapeutic areas, and effectively identifies weak binders (1%-30% hit rate), which both serve as high-quality starting points for compound design and provide extensive structural information on binding sites. The resilience of the process was demonstrated by continued screening of SARS-CoV-2 targets during the COVID-19 pandemic, including a 3-week turn-around for the main protease.

This paper describes the complete XChem process for crystal-based fragment screening, starting from applying for access and all subsequent steps to data dissemination.

In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Author Spotlight: High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography  

This protocol details high-throughput crystallization screening, ranging from the 1,536 microassay plate preparation to the end of a 6 week experimental time window. Details are included about the sample setup, the imaging obtained, and how users can perform analyses using an artificial intelligence-enabled graphical user interface to quickly and efficiently identify macromolecular crystallization conditions.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

Automated Protein Crystallization and Real-Time Data Collection

Crystallization and In Situ Room Temperature Data Collection Using the Crystallization Facility at Harwell and Beamline VMXi, Diamond Light Source

Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from crystallization plates at Diamond Light Source beamline VMXi are described. This approach enables high-quality room-temperature crystal structures to be determined from multiple crystals in a straightforward manner and provides very rapid feedback on the results of crystallization trials as well as enabling serial crystallography. The value of room temperature structures in understanding protein structure, ligand binding, and dynamics is becoming increasingly recognized in the structural biology community. This pipeline is accessible to users from all over the world with several available modes of access. Crystallization experiments that are set up can be imaged and viewed remotely with crystals identified automatically using a machine learning tool. Data are measured in a queue-based system with up to 60&#176; rotation datasets from user-selected crystals in a plate. Data from all the crystals within a particular well or sample group are automatically merged using xia2.multiplex with the outputs straightforwardly accessed via a web browser interface.

Author Spotlight: Advancing Protein Structure Analysis for Drug Development 

We present a protocol for the crystallization of proteins using the crystallization facility in the Research Complex at Harwell and subsequent in situ X-ray crystallographic data collection from crystals within the plates at Diamond&#39;s Versatile Macromolecular Crystallography in situ (VMXi) beamline. We describe sample requirements, crystallization protocols, and data collection guidelines.

The Automated Crystallography Pipelines at the EMBL HTX Facility in Grenoble

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