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://jove.unicartagenaproxy.elogim.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. 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.

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

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 &#181;m crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

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 ...

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

Fixed Target Serial Data Collection at Diamond Light Source

Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is presented with detailed step-by-step instructions, figures, and videos for smooth data collection.

We present a comprehensive guide to fixed target sample preparation, data collection, and data processing for serial synchrotron crystallography at Diamond beamline I24.

Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Bioengineering

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Biology

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current ...

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, and repair. Understanding the exact causality between genome topology and function is therefore crucial and increasingly the subject of intensive research. Chromosome conformation capture technologies (3C) allow inferring the 3D structure of chromatin by measuring the frequency of interactions between any region of the genome. Here we describe a fast and simple protocol to perform Capture Hi-C, a 3C-based target enrichment method that characterizes the allele-specific 3D organization of megabased-sized genomic targets at high-resolution. In Capture Hi-C, target regions are captured by an array of biotinylated probes before downstream high-throughput sequencing. Thus, higher resolution and allele-specificity are achieved while improving the time-effectiveness and affordability of the technology. To demonstrate its strengths, the Capture Hi-C protocol was applied to the mouse X-inactivation center (Xic), the master regulatory locus of X-chromosome inactivation (XCI).

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

This protocol describes the Capture Hi-C method used to characterize the 3D organization of megabased-sized targeted genomic regions at high-resolution, including boundaries of topologically associating domains (TADs) and long-range chromatin interactions between regulatory and other DNA sequence elements.

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, ...

The Automated Crystallography Pipelines at the EMBL HTX Facility in Grenoble

Summary

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Chapters in this video

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

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

Fixed Target Serial Data Collection at Diamond Light Source

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

The MultiBac Protein Complex Production Platform at the EMBL

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C