This work represents a large joint effort between the Diamond Light Source and the Structure Genomic Consortium. The authors would like to acknowledge Diamond&#39;s various support groups and MX group for their contribution to the automation of i04-1 beamline and for providing streamlined data collection and auto-processing pipelines, which are commonly run across all MX beamlines. They would also like to thank the SGC PX group for their resilience being the first users to test the setup and Evotec for being the first serious industrial user. This work was supported by iNEXT-Discovery (Grant 871037) funded by Horizon 2020 program of the European Commission.

The authors have no conflicts of interest to disclose.

The process outlined in this paper has been extensively tested by the user community and the adaptability of the protocols described here is key for handling the wide variety of projects typically encountered on the platform. However, a few pre-requisites of the crystal system are necessary.
For any fragment screening campaign carried out using X-ray crystallography, a reproducible and robust crystal system is critical. As the standard XChem protocol involves addition of the fragment directly to the crystal drop, optimization should focus on the number of drops containing high-quality crystals rather than the total number of crystals. If drops contain multiple crystals, then they are effectively redundant although may alleviate the harvesting process. Furthermore, transferring the crystallization protocol from the home institute to onsite facilities can be challenging. This is generally best achieved using crystal seeding to promote reproducible nucleation54, and, therefore, a good practice is for users to provide seed stocks along with their protein and crystallization solutions.
To ensure good compound solubility and support, the high soaking concentrations intended to drive binding of weak fragments, fragment libraries are provided in organic solvents, specifically DMSO and ethylene glycol. Provision of two different solvents gives users an alternative for crystals which do not tolerate DMSO at all, or where it occludes the binding of fragments in a site of interest. Users can supply alternative libraries in aqueous buffer: compounds will dispense well provided they are completely dissolved and formatted in plates compatible with the liquid dispensing robot.
For projects where it is not possible to find an appropriate organic solvent that would both solubilize the library and be tolerated by the crystal system, an alternative procedure is to use dried compounds as established at BESSY55.
In the community, there is a long-standing question about being able to soak compounds into crystals grown in crystallization conditions containing high salt concentrations. Practically, more precipitation of the compounds and rapid formation of salt crystals at the harvesting stage is observed, which is reduced by applying a humid environment around the harvesting area. Generally, screening campaigns in crystal systems from high salt crystallization conditions give a comparable hit rate to low salt conditions.
The initial stages of the XChem process (solvent tolerance testing and pre-screen) are relatively small-scale and quick experiments but allow clear go/no go decision for the project. Most painfully, alternative crystal systems will need to be found if neither solvent is tolerated, or the pre-screen results in a very low hit rate. In contrast, if they are successful, the results directly inform the soaking condition to use for the screening experiment, and the best strategy for data collection. Since quality of the data, especially the resolution, will affect the quality of the electron density for hit identification and analysis, the aim is to soak at the highest possible compound concentration that does not have a deleterious effect on diffraction quality (with the majority of datasets (~80%) diffracting to a resolution of 2.8 &#197; or better).
The data analysis process is streamlined within XChemExplorer, which relies on the PanDDA software for the detection of weak binders and allows users to quickly visualize and review the outcomes of the screening campaign. XChemExplorer imports data processing results from the packages available at Diamond (DIALS16, autoPROC30, STARANISO31, and Xia214) with resolution limits determined by the standard method for each package (i.e., CC1/2 = 0.3). By default, dataset selection is based on a score calculated from I/sigI, completeness, and a number of unique reflections, but specific processing results can be selected for use both globally or for individual samples25. Data is also excluded from analysis by PanDDA based on criteria including resolution, Rfree, and difference in unit cell volume between reference and target data (defaults are 3.5 &#197;, 0.4, and 12% respectively), so that poorly diffracting, mis-centered, or mis-indexed crystals do not affect the analysis.
The PanDDA algorithm takes advantage of the substantial number of datasets collected during a fragment campaign to detect partial occupancy ligands that are not visible in standard crystallographic maps. Initally, PanDDA uses data collected during the solvent tolerance testing and pre-screen steps to prepare an average density map which is then used to create a ground-state model. As this model will be used for all subsequent analysis steps, it is vital that it accurately represents the un-liganded protein under the conditions used for the fragment screen. PanDDA then uses a statistical analysis to identify bound ligands, generating an event map for the bound state of the crystal. An event map is generated by subtracting the unbound fraction of the crystal from the partial-occupancy dataset and presents what would be observed if the ligand was bound at full occupancy. Even fragments that appear clear in conventional 2mFo-DFc maps might be mismodeled if the event maps are not consulted32. While PanDDA is a powerful method for identifying datasets that differ from the average maps (which is usually indicative of fragment binding) and metrics such as RSCC, RSZD, B-factor ratio, and RMSD during refinement are provided for the users benefit, the user is ultimately responsible for deciding whether the observed density accurately depicts the expected ligand and the most suitable conformation.
Following data analysis and refinement, it is possible for all users to simultaneously deposit multiple structures in the Protein Data Bank (PDB) using XChemExplorer. For each fragment-screen, two group depositions are made. The first deposition contains all fragment-bound models, with coefficients for calculating PanDDA event maps included in MMCIF files. The second deposition provides the accompanying ground-state model, along the measured structure factors of all datasets of the experiment: this data can be used to reproduce the PanDDA analysis, and for developing future algorithms. As for the structures of the hits, when fragment occupancy is low, refinement is better behaved if models are a composite of the ligand-bound and confounding ground-state structures32; nevertheless, the practice is to deposit only the bound-state fractions, since the full composite models are in general complex and difficult to interpret. As a result, some quality indicators recalculated by the PDB (in particular, R/Rfree) are sometimes slightly elevated. It is also possible to provide all raw data using platforms such as Zenodo56, although this is not currently supported by the XChem pipeline.
Overall, since its operation in 2016, fragment ligands could be identified in over 95% of the targets using this procedure. Experience from the many projects that XChem has supported was distilled into best practice for crystal preparation33, while a fragment library was evolved that implemented the poised concept for aiding fragment progression29, also helping establish the practice of making library composition public. The platform has demonstrated the importance of well-maintained infrastructure and documented processes, detailed here, and made it possible to evaluate other fragment libraries57,58, to compare libraries48, and to inform the design of the collaborative EUOpenscreen-DRIVE library59,60.

Fragment-Based Drug Discovery (FBDD) is a widely-used strategy for lead discovery, and since its emergence 25 years ago, it has delivered four drugs for clinical use and more than 40 molecules have been advanced to clinical trials1,2,3. Fragments are small chemical entities usually with a molecular weight of 300 Da or less. They are selected for their low chemical complexity, which provide good starting points for development of highly ligand efficient inhibitors with excellent physicochemical properties. Their size means that they sample the binding landscape of proteins more thoroughly than libraries of larger drug- or lead-like compounds, and thus also reveal hot spots and putative allosteric sites. Combined with structural information, fragments provide a detailed map of the potential molecular interactions between protein and ligand. Nevertheless, reliably detecting and validating those entities, which tend to bind weakly to the target protein, requires an array of robust and sensitive biophysical screening methods such as Surface Plasmon Resonance (SPR), Nuclear Magnetic Resonance (NMR), or Isothermal Titration Calorimetry (ITC)4,5.
X-ray crystallography is an essential part of the FBDD toolkit: it is sensitive enough to identify weak binders and directly yields structural information about the interactions at a molecular level. It is complementary to other biophysics screens and usually essential for progressing fragment hits to lead compounds; it requires high quality crystal systems, meaning that crystallization is highly reproducible, and crystals ideally diffract to better than 2.8 &#197; resolution.
Historically, it has been very difficult to use crystallography as primary fragment screen6,7,8, whether in academia or in industry. In contrast, synchrotrons achieved order of magnitude improvements in robotics, automation9,10,11 and detector technology12,13, and combined with equally accelerated computing power and algorithms of data processing14,15,16, complete diffraction datasets can be measured in seconds and large numbers of them entirely unattended, as pioneered at LillyCAT7 and later MASSIF17,18 (European Synchrotron Radiation Facility (ESRF)). This led synchrotrons to develop highly streamlined platforms to make crystal-based fragment screening as primary screen accessible to a wide user community (XChem at Diamond; CrystalDirect at EMBL/ESRF19; BESSY at Helmholtz-Zentrum Berlin20; FragMax at MaxIV21).
This paper documents the protocols that constitute the XChem platform for fragment screening by X-ray crystallography, from sample preparation to the final structural results of 3D-modeled hits. The pipeline (Figure 1) required developing new approaches to crystal identification22, soaking23, and harvesting24, as well as data management software25 and an algorithmic approach to identifying fragments26 that is now widely used in the community. The crystal harvesting technology is now sold by a vendor (see Table of Materials), and the open availability of the tools has allowed other synchrotrons to adapt them to set up equivalent platforms21. Ongoing projects address data analysis, model completion, and data dissemination through the Fragalysis platform27. The sample preparation laboratory is adjacent to beamline I04-1, simplifying the logistics of transferring hundreds of frozen samples to the beamline and dedicated beamtime on I04-1 allows rapid X-ray feedback to guide the campaign.
XChem is an integral part of Diamond&#39;s user program, with two calls per year (early April and October). The peer-review process has been refined in consultation with experts in drug discovery from Academia and Industry. Along with a strong science case, the proposal process28 requires applicants to self-assess not only the readiness of the crystal system, but also their expertise in biochemical and orthogonal biophysical methods and capacity to progress screening hits through follow-up chemistry. The modes of access have also evolved to accommodate the multidisciplinary user community:
Tier 1 (single project) is for projects at the exploratory stage and hit validation tools (biophysics or biochemical tools) and follow-up strategies need not be in place. If accepted, the project is granted a reduced number of beamtime shifts, enough for proof of concept.
Tier 2 (single project) is for well-validated projects and requires downstream tools and follow-up strategies to be in place. If accepted, the project is allocated enough beamtime for a full fragment screening campaign. Single projects (Tier 1 or Tier 2) are to be completed within the 6 months of the allocation period (either April to September or October to March).
Block Allocation Group (BAG) is for a consortia of groups and projects, where a robust target selection and prioritization process is in place within the BAG, along with a clear follow-up pipeline. BAGs must have at least one fully XChem trained expert (superuser), who coordinates their activities with Diamond staff and trains the BAG members. The allocated number of beamtime shifts is defined by the number of scientifically strong projects in the BAG and is re-evaluated per allocation period based on the BAG&#39;s report. The access is available for 2 years.
The XChem experiment is divided into three stages, with a decision point for each of them: solvent tolerance test, pre-screen, and main screen (Figure 2). The solvent tolerance test helps define the soaking parameters, the amount of solvent (DMSO, ethylene glycol, or other cryoprotectants if needed) the crystal system can tolerate and for how long. Solvent concentrations typically range from 5%-30% over at least two time points. Diffraction data is collected and compared to the base diffraction of the crystal system; this will determine the soaking parameters for the following stage. For the pre-screen, 100-150 compounds are soaked using the conditions determined in the solvent test, and its purpose is to confirm that the crystals can tolerate the compounds in those conditions. If needed, the cryoprotectant is subsequently added to the drops already containing the fragments. The success criteria are that 80% or more of the crystals survive well enough to yield diffraction data of good and consistent quality; if this fails, soaking conditions are usually revised by altering the soak time or solvent concentration. Following a successful pre-screen, the rest of the compounds chosen for the experiment can be set up using the final parameters.
The DSI-poised library (see Table of Materials) was purposely designed to allow rapid follow-up progression using poised chemistry29 and has been the facility&#39;s workhorse library. It is available to users at a concentration of 500 mM in DMSO. Academic users can also access other libraries provided by collaborators (over 2,000 compounds in total) at concentrations of 100-500 mM in DMSO (a full list can be found on the website28). Much of the overall collection is also available in ethylene glycol, for crystal systems that do not tolerate DMSO. Users can also bring their own libraries, provided they are in plates compatible with the acoustic liquid handling system (see Table of Materials).
For all three steps of the experiment (solvent characterization, pre-screen or full screen), the following sample preparation procedures are identical (Figure 3): selection of the compound dispensing location through imaging and targeting of crystallization drops with TeXRank22; dispensing into drops using the acoustic liquid dispensing system for both solvent and compounds23; efficient harvesting of the crystals using the Crystal shifter24; and upload of sample information into the beamline database (ISPyB). The current interface for experiment design and execution is an Excel-based application (SoakDB), which generates the necessary input files for the different equipment of the platform, and tracks and records all results in an SQLite database. Barcode scanners are used at various stages throughout the process to help track samples and this data is added to the database.
Diffraction data are collected in unattended mode using dedicated beamtime on beamline I04-1. Two centering modes are available, namely, optical and X-ray based17. For needle and rod-shaped crystals, X-ray centering is advised, whereas chunkier crystals generally support optical mode, which is faster and, therefore, allows for more samples to be collected in the allotted beamtime. Depending on the resolution of the crystals (established before entering the platform) data collection can either be 60 s or 15 s total exposure. Data collection during the solvent test stage usually informs which combination will work best with the performance of beamline I04-1.
The large volume of data analysis is managed through XChemExplorer (XCE)25, which can also be used to launch the hit identification step using PanDDA26. XCE is a data management and workflow tool that supports large-scale analysis of protein-ligand structures (Figure 4); it reads any of the auto-processing results from data collected at Diamond Light Source (DIALS16, Xia214, AutoPROC30, and STARANISO31) and auto-selects one of the results based on data quality and similarity to a reference model. It is important that the model is representative of the crystal system used for XChem screening, and must include all waters or other solvent molecules, as well as all co-factors, ligands, and alternative conformations visible in crystals soaked with solvent only. The quality of this reference model will directly impact the amount of work required during the model building and refinement stage. PanDDA is used to analyze all the data and identify binding sites. It aligns structures to a reference structure, calculates the statistical maps, identifies events, and calculates event maps26,32. In the PanDDA paradigm, it is neither necessary nor desirable to build the full crystallographic model; what must be modeled is only the view of the protein where a fragment is bound (the bound-state model), so the focus need be only on building the ligand and surrounding residues/solvent molecules according to the event map32.

<table><tbody><tr><td>DSI-poised library</td><td>Enamine</td><td>DSI-896</td><td>fragment library</td></tr><tr><td>Echo 550 and 650 series</td><td>Beckman-Coulter</td><td></td><td>acoustic dispensing system</td></tr><tr><td>Echo microplates</td><td>Beckman-Coulter</td><td>001-12380; 001-8768; 001-6025</td><td>1536-well and 384-well microplates</td></tr><tr><td>Shifter</td><td>Oxford Lab Technology</td><td></td><td>harvesting device</td></tr><tr><td>Microplate centrifuge with a swing-out rotor</td><td>Sigma</td><td>model 11121</td><td>microplate centrifuge</td></tr><tr><td>3-drops crystallisation plates</td><td>Swissci</td><td>3W96T-UVP</td><td>Crystallisation plates</td></tr><tr><td>Formulatrix plate imager and Rockmaker software</td><td>Formulatrix</td><td></td><td>Crystallisation plates imaging device</td></tr></tbody></table>

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.

The XChem pipeline for fragment screening by X-Ray crystallography has been extensively streamlined, enabling its uptake by the scientific community (Figure 5). This process has been validated on over 150 of screening campaigns with a hit rate varying between 1% and 30%47,48,49,50,51,52&#160;and by many repeat users. Crystal systems that are not suitable (low resolution, inconsistent in crystallization or in diffraction quality) or cannot tolerate either DMSO or ethylene glycol are eliminated early in the process, saving time, effort, and resource. Successful campaigns provide a three-dimensional map of potential interaction sites on the target protein; a typical outcome is the XChem screen of the main protease of SARS-CoV-2 (Figure 6). Typically, fragment hits are found in: (a) known sites of interest, such as enzyme active sites and sub-pockets48; (b) putative allosteric sites, for example, in protein-protein interactions53; (c) crystal packing interfaces, generally considered as false positives (Figure 6). This structural data generally provides a basis for merging, linking, or growing fragment hits into lead-like small molecules1,3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62414/62414fig01.jpg" /> 
Figure 1: The XChem pipeline. The platform is represented schematically from project proposal through sample preparation, data collection, and hit identification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62414/62414fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62414/62414fig02.jpg" /> 
Figure 2: Screening strategy. The workflow indicates the purpose of each milestone, the experiment&#39;s requirements, and the decision points. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62414/62414fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62414/62414fig03.jpg" /> 
Figure 3: Sample preparation workflow. Critical steps for the sample preparation are represented with information from each step being recorded in an SQLite database. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62414/62414fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62414/62414fig04.jpg" /> 
Figure 4: Data analysis using XCE. Critical steps in the data analysis are represented by a workflow diagram with the relevant software packages. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62414/62414fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62414/62414fig05.jpg" /> 
Figure 5: Evolution of the XChem user program: The chart demonstrates the uptake and consolidation of the user program from 2015 through to 2019 with the creation of BAGs in 2019 and the resilience of the platform through the COVID-19 pandemic in 2020. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62414/62414fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62414/62414fig06.jpg" /> 
Figure 6: Representative results of XChem fragment screen. SARS-CoV2 main protease (Mpro) dimer is represented in surface with active site hits shown in yellow, putative allosteric hits shown in magenta, and surface/crystal-packing artefacts shown in green. The figure was made using Chimera and Mpro PDB entries from group deposition G_1002156. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62414/62414fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source at JoVE.com

Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source

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 ...

achieving-efficient-fragment-screening-at-xchem-facility-at-diamond

Nanyang Technological University

Research

JoVE Journal

Chemistry

5.1K Views.  Diamond Light Source Ltd, Harwell Science and Innovation Campus. De XChem-faciliteit bij Diamond Light Source biedt routinematige, grootschalige screening van kristalgrafische fragmenten, ter ondersteuning van het hele proces van kristal tot afzetting. Röntgenkristallografie is een essentieel onderdeel van de FBDD-toolkit. Het is gevoelig genoeg om zwakke bindmiddelen te identificeren en levert direct structurele informatie op over de interacties op moleculair niveau.Fragment based drug discovery is een veelgebruikte strategie voor lead discovery. ...

Video: Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Biochemistry

Synchrotron X-ray Microdiffraction and Fluorescence Imaging of Mineral and Rock Samples

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder microdiffraction two-dimensional (2D) maps at beamline 12.3.2 of the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. Measurements can be performed on any sample that is less than 10 cm x 10 cm x 5 cm, with a flat exposed surface. The experimental geometry is calibrated using standard materials (elemental standards for XRF, and crystalline samples such as Si, quartz, or Al2O3 for diffraction). Samples are aligned to the focal point of the x-ray microbeam, and raster scans are performed, where each pixel of a map corresponds to one measurement, e.g., one XRF spectrum or one diffraction pattern. The data are then processed using the in-house developed software XMAS, which outputs text files, where each row corresponds to a pixel position. Representative data from moissanite and an olive snail shell are presented to demonstrate data quality, collection, and analysis strategies.

We describe a beamline setup meant to carry out rapid two-dimensional x-ray fluorescence and x-ray microdiffraction mapping of single crystal or powder samples using either Laue (polychromatic radiation) or powder (monochromatic radiation) diffraction. The resulting maps give information about strain, orientation, phase distribution, and plastic deformation.

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder ...

Engineering

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 ...

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 ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...

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 ...

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 &#197;. 
To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. 
Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

Here, we present a protocol for cryogenic sample preparation and transfer of crystals into the vacuum endstation on beamline I23 at Diamond Light Source, for long-wavelength macromolecular X-ray crystallography experiments.

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident high-energy electron beam is rocked with the submicrometric pivot point fixed on a specimen. This method enables us to quantitatively derive the site occupancies and site-dependent chemical information of impurities or intentionally doped functional elements in a specimen, using energy-dispersive X-ray spectroscopy and electron energy-loss spectroscopy attached to a scanning transmission electron microscope, which is of significant interest to current materials science, particularly related to nanotechnologies. This scheme is applicable to any combination of elements even when the conventional Rietveld analysis by X-ray or neutron diffraction occasionally fails to provide the desired results because of limited sample sizes and close scattering factors of neighboring elements in the periodic table. In this methodological article, we demonstrate the basic experimental procedure and analysis method of the present beam-rocking microanalysis.

We provide a general outline of quantitative microanalysis methods for estimating the site occupancies of impurities and their chemical states by taking advantage of electron-channeling phenomena under incident electron beam-rocking conditions, which reliably extract information from minority species, light elements, oxygen vacancies, and other point/line/planar defects.

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident ...

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.

Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source

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