Crystal Hits Research Articles

CHiMP: deep-learning tools trained on protein crystallization micrographs to enable automation of experiments

A group of three deep-learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate), were created for analysis of micrographs of protein crystallization experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case and masks of crystallization droplets in addition to crystals in the second case, allowing the positions and sizes of these entities to be recorded. The creation of these tools used transfer learning, where weights from a pre-trained deep-learning network were used as a starting point and repurposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS, where they have the potential to absolve the need for any user input, both for monitoring crystallization experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment-based drug-discovery screening platform, also at DLS, to allow the automatic targeting of acoustic compound dispensing into crystallization droplets.

Ethoxylate Polymer-Based 96-Well Screen for Protein Crystallization

Crystallization is the limiting step in X-ray structure determination of biological macromolecules. As crystallization experiments can be largely automatized, the diversity of precipitant solutions is often the determinant factor to obtain crystals of high quality. Here, we introduce a 96-well screening kit of crystallization conditions, centered on three ethoxylate-based organic polymers as precipitants and various additional compounds to promote crystal formation. This crystallization screen was tested on various non-standard proteins from bacteria and archaea. Structure determination succeeded for seven out of thirteen targets based on crystals that frequently diffracted to a higher resolution than those obtained with commercially available screening kits. Crystallization hits were rarely similar among the three ethoxylate-based organic polymers and, in comparison, with already available crystallization screens. Hence, the presented crystallization screen is an efficient tool to complement other screens and increase the likelihood of growing crystals suitable for X-ray structure determination.

Structure-guided computational insecticide discovery targeting β-N-acetyl-D-hexosaminidase of Ostrinia furnacalis

Ostrinia furnacalis is a species of moth in the Crambidae family that is harmful to maize and other corn crops in Southeast Asia and the Western Pacific regions. Ostrinia furnacalis causes devastating losses to economically important corn fields. The β-N-acetyl-D-hexosaminidase is an essential enzyme in O. furnacalis and its substrate binding +1 active site is different from that of the plants and humans β-N-acetyl-D-hexosaminidases. To develop environment-friendly insecticides against OfHex1, we conducted structure-guided computational insecticide discovery to identify potential inhibitors that can bind the active site and inhibit the substrate binding and activity of the enzyme. We adopted a three-pronged strategy to conduct virtual screening using Glide and virtual screening workflow (VSW) in Schrödinger Suite-2022-3, against crystal structures of OfHex1 (PDB Id:3NSN), its homologue in humans (PDB Id: 1NP0) and Alphafold model of β-N-acetyl-D-hexosaminidase from Trichogramma pretiosum, an egg parasitoid that protects the crops from O. furnacalis. A library of 20,313 commercially available and “insecticide-like” compounds was extracted from published literature. LigPrep enabled 44,943 ready-to-dock conformers generation. Glide docking revealed 18 OfHex1-specific hits that were absent in human and T. pretiosum screens. Reference docking was conducted using inhibitors/natural ligands in the crystal structures and hits with better docking scores than the reference were selected for MD simulations using Desmond to understand the stability of hit-target interactions. We noted five compounds that bound to OfHex1 TMX active-site based on their docking scores, consistent binding as noted by MD simulations and their insecticide/pesticide likeliness as noted by the Comprehensive Pesticide Likeness Analysis. Communicated by Ramaswamy H. Sarma

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography.

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

20 years of crystal hits: progress and promise in ultrahigh-throughput crystallization screening.

Diffraction-based structural methods contribute a large fraction of the biomolecular structural models available, providing a critical understanding of macromolecular architecture. These methods require crystallization of the target molecule, which remains a primary bottleneck in crystal-based structure determination. The National High-Throughput Crystallization Center at Hauptman-Woodward Medical Research Institute has focused on overcoming obstacles to crystallization through a combination of robotics-enabled high-throughput screening and advanced imaging to increase the success of finding crystallization conditions. This paper will describe the lessons learned from over 20 years of operation of our high-throughput crystallization services. The current experimental pipelines, instrumentation, imaging capabilities and software for image viewing and crystal scoring are detailed. New developments in the field and opportunities for further improvements in biomolecular crystallization are reflected on.

A drug-discovery-oriented non-invasive protocol for protein crystal cryoprotection by dehydration, with application for crystallization screening.

In X-ray macromolecular crystallography, cryoprotection of crystals mounted on harvesting loops is achieved when the water in the sample solvent transitions to vitreous ice before crystalline ice forms. This is achieved by rapid cooling in liquid nitro-gen or propane. Protocols for protein crystal cryoprotection are based on either increasing the environmental pressure or reducing the water fraction in the solvent. This study presents a new protocol for cryoprotecting crystals. It is based on vapour diffusion dehydration of the crystal drop to reduce the water fraction in the solvent by adding a highly concentrated salt solution, 13 M potassium formate (KF13), directly to the reservoir. Several salt solutions were screened to identify KF13 as optimal. Cryoprotection using the KF13 protocol is non-invasive to the crystal, high throughput and easy to implement, can benefit diffraction resolution and ligand binding, and is very useful in cases with high redundancy such as drug-discovery projects which use very large compound or fragment libraries. An application of KF13 to discover new crystal hits from clear drops of equilibrated crystallization screening plates is also shown.

Pembuatan dan Karakterisasi Rochelle Salt Crystal

Rochelle Salt is the first synthesis Crystal made by Pierre Siegnette La Rocchelle in France. Rochelle Salt have several characteriztion one of them is piezoelectric effect where the crystal can change the kinetic energy to electrical energy instead this is the one of rewenable source altenative energy. Rochelle Salt can be made with tartar cream and washing soda reacted with distilled water as a solvent. The solution is reacted until there is no more reaction or supersaturation condition. Wait the solution for 2 days until the crystal seed appears. The crystal growth using the method of growing crystal in solution for 1 month until the crystal bigger enough for research to find the piezoelectric effect. Based on the result that have been obtained, Rochelle Salt have piezoelectric effect. Crystal hit using with wooden hitter and metal hitter, the voltage is increase when the crystal is hit by hitter. The piezoelectric also disappear along with time.

Effect of X-ray free-electron laser-induced shockwaves on haemoglobin microcrystals delivered in a liquid jet

X-ray free-electron lasers (XFELs) enable obtaining novel insights in structural biology. The recently available MHz repetition rate XFELs allow full data sets to be collected in shorter time and can also decrease sample consumption. However, the microsecond spacing of MHz XFEL pulses raises new challenges, including possible sample damage induced by shock waves that are launched by preceding pulses in the sample-carrying jet. We explored this matter with an X-ray-pump/X-ray-probe experiment employing haemoglobin microcrystals transported via a liquid jet into the XFEL beam. Diffraction data were collected using a shock-wave-free single-pulse scheme as well as the dual-pulse pump-probe scheme. The latter, relative to the former, reveals significant degradation of crystal hit rate, diffraction resolution and data quality. Crystal structures extracted from the two data sets also differ. Since our pump-probe attributes were chosen to emulate EuXFEL operation at its 4.5 MHz maximum pulse rate, this prompts concern about such data collection.

Belle II electromagnetic calorimeter and its performance during early SuperKEKB operation

The Belle II experiment at the SuperKEKB accelerator is the new-generation intensity frontier electron-positron colliding beam experiment. The electromagnetic calorimeter consisting of 8736 CsI(Tl) crystals with their PIN-photodiode readout, has been inherited from the Belle experiment. In order to achieve high rate capability and immunity against beam background, the electronics has been upgraded to feature waveform sampling readout to provide not only energy deposition but also timing for each crystal hit. Design and construction of the upgraded electronics are reviewed and the performance in the early Belle II physics runs is reported. We also present the first application of calorimeter pulse shape discrimination at a B-Factory experiment. This novel technique, enabled by the new waveform sampling electronics, allows for discrimination between electromagnetic and hadronic showers, leading to significant improvements in kaon-long vs. photon separation and charged particle identification.

Tailored Suits Fit Better: Customized Protein Crystallization Screens

The major bottleneck in macromolecular crystallography is the production of well-diffracting crystals. To increase the crystallization success, we developed a crystallization strategy customizable for each protein and tested it on selected model and in-house non-yet-crystallized proteins. The underlying concept is to use in crystallization cocktails compounds that increase the thermal stability of the protein. This customized strategy yielded not only twice as many crystal hits as the standard approach but also crystal hits for two additional proteins compared to the benchmark workflow. The inherently simple design and the modular and flexible nature of the platform makes it easy to modify, further develop, and optimize individual steps. The information gained can also be used to increase the monodispersity and stability of proteins, improving in this way their amenability for biochemical studies and eventually derive a possible function of not yet annotated proteins.

3D printed droplet generation devices for serial femtosecond crystallography enabled by surface coating.

The role of surface wetting properties and their impact on the performance of 3D printed microfluidic droplet generation devices for serial femtosecond crystallography (SFX) are reported. SFX is a novel crystallography method enabling structure determination of proteins at room temperature with atomic resolution using X-ray free-electron lasers (XFELs). In SFX, protein crystals in their mother liquor are delivered and intersected with a pulsed X-ray beam using a liquid jet injector. Owing to the pulsed nature of the X-ray beam, liquid jets tend to waste the vast majority of injected crystals, which this work aims to overcome with the delivery of aqueous protein crystal suspension droplets segmented by an oil phase. For this purpose, 3D printed droplet generators that can be easily customized for a variety of XFEL measurements have been developed. The surface properties, in particular the wetting properties of the resist materials compatible with the employed two-photon printing technology, have so far not been characterized extensively, but are crucial for stable droplet generation. This work investigates experimentally the effectiveness and the long-term stability of three different surface treatments on photoresist films and glass as models for our 3D printed droplet generator and the fused silica capillaries employed in the other fluidic components of an SFX experiment. Finally, the droplet generation performance of an assembly consisting of the 3D printed device and fused silica capillaries is examined. Stable and reproducible droplet generation was achieved with a fluorinated surface coating which also allowed for robust downstream droplet delivery. Experimental XFEL diffraction data of crystals formed from the large membrane protein complex photosystem I demonstrate the full compatibility of the new injection method with very fragile membrane protein crystals and show that successful droplet generation of crystal-laden aqueous droplets intersected by an oil phase correlates with increased crystal hit rates.

Organizing a crystallization laboratory

Managing chemical stocks and samples in any laboratory is an arduous task; in a crystallization laboratory this is particularly burdensome, given the need for many stocks to facilitate optimization of crystal hits obtained from screening experiments. Although inventory management is widespread in retail and other arenas, most small academic laboratories do not adopt formal stock management systems. Without an overarching system for handling stocks and samples, problems such as stock duplication, inappropriate stock storage and insufficient labelling are rife. Two applications have been developed in the Collaborative Crystallization Centre, the first of which manages the hundreds of stocks used for crystallization, and a second which manages protein (and other) samples stored in the 193 K freezer. Both applications are built around a simple database, with a Python front end that allows samples or stocks to be scanned in or out. Information from a decade of crystallization stock usage allows a good estimation of what chemicals are used (and in what quantities) in a crystallization laboratory.

Hit detection in serial femtosecond crystallography using X-ray spectroscopy of plasma emission.

Serial femtosecond crystallography is an emerging and promising method for determining protein structures, making use of the ultrafast and bright X-ray pulses from X-ray free-electron lasers. The upcoming X-ray laser sources will produce well above 1000 pulses per second and will pose a new challenge: how to quickly determine successful crystal hits and avoid a high-rate data deluge. Proposed here is a hit-finding scheme based on detecting photons from plasma emission after the sample has been intercepted by the X-ray laser. Plasma emission spectra are simulated for systems exposed to high-intensity femtosecond pulses, for both protein crystals and the liquid carrier systems that are used for sample delivery. The thermal radiation from the glowing plasma gives a strong background in the XUV region that depends on the intensity of the pulse, around the emission lines from light elements (carbon, nitrogen, oxygen). Sample hits can be reliably distinguished from the carrier liquid based on the characteristic emission lines from heavier elements present only in the sample, such as sulfur. For buffer systems with sulfur present, selenomethionine substitution is suggested, where the selenium emission lines could be used both as an indication of a hit and as an aid in phasing and structural reconstruction of the protein.

A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase.

Random microseed matrix screening (rMMS), in which seed crystals are added to random crystallization screens, is an important breakthrough in soluble protein crystallization that increases the number of crystallization hits that are available for optimization. This greatly increases the number of soluble protein structures generated every year by typical structural biology laboratories. Inspired by this success, rMMS has been adapted to the crystallization of membrane proteins, making LCP seed stock by scaling up LCP crystallization conditions without changing the physical and chemical parameters that are critical for crystallization. Seed crystals are grown directly in LCP and, as with conventional rMMS, a seeding experiment is combined with an additive experiment. The new method was used with the bacterial integral membrane protein OmpF, and it was found that it increased the number of crystallization hits by almost an order of magnitude: without microseeding one new hit was found, whereas with LCP-rMMS eight new hits were found. It is anticipated that this new method will lead to better diffracting crystals of membrane proteins. A method of generating seed gradients, which allows the LCP seed stock to be diluted and the number of crystals in each LCP bolus to be reduced, if required for optimization, is also demonstrated.

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.

Crystallization of P-type ATPases by the High Lipid-Detergent (HiLiDe) Method.

Determining structures of membrane proteins remains a significant challenge. A technique utilizing high lipid-detergent concentrations ("HiLiDe") circumvents the major bottlenecks of current membrane protein crystallization methods. During HiLiDe, the protein-lipid-detergent ratio is varied in a controlled way in order to yield initial crystal hits, which may be subsequently optimized by variation of the crystallization conditions and/or utilizing secondary detergents. HiLiDe preserves the advantages of classical lipid-based methods, yet is compatible with both the vapor diffusion and batch crystallization techniques. The method has been applied with particular success to P-type ATPases.

Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams.

High-resolution structural information was obtained from lysozyme microcrystals (20 µm in the largest dimension) using raster-scanning serial protein crystallography on micro- and nano-focused beamlines at the ESRF. Data were collected at room temperature (RT) from crystals sandwiched between two silicon nitride wafers, thereby preventing their drying, while limiting background scattering and sample consumption. In order to identify crystal hits, new multi-processing and GUI-driven Python-based pre-analysis software was developed, named NanoPeakCell, that was able to read data from a variety of crystallographic image formats. Further data processing was carried out using CrystFEL, and the resultant structures were refined to 1.7 Å resolution. The data demonstrate the feasibility of RT raster-scanning serial micro- and nano-protein crystallography at synchrotrons and validate it as an alternative approach for the collection of high-resolution structural data from micro-sized crystals. Advantages of the proposed approach are its thriftiness, its handling-free nature, the reduced amount of sample required, the adjustable hit rate, the high indexing rate and the minimization of background scattering.

Structure determination from in situ diffraction with the Rigaku PlateMate

As crystallographers face increasing problems with crystallizing new proteins, in-situ screening in crystallization trays at room temperature is experiencing a renaissance. It saves a lot of time when screening large numbers of crystallization hits and it helps avoid crystal damage caused by human manipulation error (harsh manual handling, bad freezing) or changes in crystal properties (dehydration, wrong cryo-conditions). In certain cases, it is also possible to go beyond screening and collect enough data for structure solution, especially on an X-ray home source where a less intense beam helps minimize the devastations of radiation damage occurring at room temperature. The Rigaku PlateMate has proved itself as an efficient and easy-to-use in-situ screening tool on the field for the past two years. It is as easily mounted on a goniometer as a regular goniometer head and thanks to a plate adapter with SBS footprints, it accommodates most 96-wells plate types, from sitting and hanging drop to LCP plates. In addition, thanks to its narrow dimensions and aided by software to prevent collisions with the detector and the crystal viewing camera, the PlateMate can be used to easily collect data from crystals in situ. In this work, we present structure solution results obtained from data collected with the PlateMate on crystals from various proteins (native crystals or containing gold or iodine) and using one or multiple crystals to make up a complete data set.

"How to use ""random"" microseeding before you get your first crystals"

Random Microseed Matrix-Screening (rMMS), where seed crystals are added automatically to random crystallization screens, is a significant recent breakthrough in protein crystallization [1]. One industrial group used the method to solve 38 out of 70 structures generated in a three year period, finding particular success with antibody complexes [2]. rMMS not only produces more hits, it also generates better-diffracting crystals - because crystals are more likely to grow in the metastable zone. This assumes, however, that you have some initial crystal hits to make a seed stock from. This presentation will look at unusual methods of nucleation, including cross-seeding [3], heterogeneous nucleation, and nucleation with precipitants [4].

AGIPD detector for Serial Femtosecond Crystallography Apparatus at European XFEL

The European X-ray Free-Electron Laser (XFEL.EU) [1] will provide ultra-short, highly-intense, coherent x-ray pulses at an unprecedented repetition rate, transforming experiments in many scientific areas, including serial femtosecond crystallography (SFX). For the purpose of SFX experiments at the XFEL.EU, a dedicated endstation is being developed to be installed within the Single Particles, Clusters and Biomolecules (SPB) instrument [2]. The setup will refocus the beam spent by SPB into a second interaction region, thereby enabling two parallel experiments. In order to overcome various challenges in XFEL crystallography, and to optimize the output for SFX experiments at XFEL.EU, the Adaptive Gain Integrating Pixel Detector (AGIPD) [3] is currently under development and is to be implemented in the SPB instrument, including a 4 Megapixel version for the SFX apparatus. The AGIPD is a hybrid-pixel detector with pixels of 200 x 200 micron^2 each. The gain of each single pixel dynamically and independently adapts to the incoming signal. Thus, diffraction patterns of high dynamic range can be recorded, with the measured signal within a single data frame ranging from single photons and up to 1e+4 photons at 12 keV. Moreover, the AGIPD is designed to store over 350 data frames from successive pulses prior to digitization and read-out, thereby enabling operation at the European XFEL with its challenging repetition rate with 10 Hz pulse trains and a 4.5 MHz intra-train repetition rate. Furthermore, the incorporation of a veto system in AGIPD will allow one to potentially store only the frames that contain diffraction data from actual crystal hits, which ultimately increases the efficiency of the detector and DAQ systems dramatically. In the present work, we will review the design of the 4Mpix AGIPD for the SFX apparatus and discuss simulations and tests of its expected performance under the conditions foreseen for SFX experiments at the XFEL.EU.