The video details a protocol for analyzing a thawed sample using centrifugation, microscopy, and imaging techniques over six weeks. It focuses on identifying crystal growth and precipitation through automated plate imaging, utilizing brightfield, sonic, and UV-TPEF imaging to characterize the samples' properties and monitor changes.

protein sample setup and crystallization

蛋白质结晶的高通量筛选

Even in an age of tremendous progress in structural biology methods, X-ray crystallography continues to be a dependable and popular method for generating high-quality structural models of macromolecules. Over 85% of all three-dimensional structural models deposited to the Protein Data Bank (PDB) are from crystal-based structural methods (as of January, 2023).1 Furthermore, X-ray crystallography remains indispensable for solving protein-ligand structures, a crucial component of the drug discovery and development process2. Despite protein crystallization having remained the dominant structural biology technique for over half a century, methods to predict crystallization likelihood based on physical properties3 or sequence4,5 are still in their infancy.
The prediction of crystallization conditions is even more obscure; limited progress has been made to predict likely crystallization conditions even for model proteins6,7. Other studies have attempted to identify crystallization conditions based on protein homology and conditions mined from the PDB8,9,10. The predictive power to be found in the PDB is limited, however, as only the final, successful crystallization conditions are deposited, which, by necessity, misses the often extensive optimization experiments required to fine-tune crystal growth. Further, many PDB entries lack metadata containing these details, including the cocktail formulas, crystallization format, temperature, and time to crystallize11,12. Therefore, for many proteins of interest, the most accessible way to determine the crystallization conditions is experimentally, using as many conditions as possible across a wide range of chemical possibilities.
Several approaches to make crystallization screening as fruitful and thorough as possible have been explored to great effect, including sparse matrices13, incomplete factorial screening14, additives15,16, seeding17, and nucleating agents18. The National HTX Center at Hauptman-Woodward Medical Research Institute (HWI) has developed an efficient pipeline for crystallization screening using the microbatch-under-oil approach19, which utilizes automated liquid handling and imaging modalities to streamline the identification of initial crystallization conditions using comparatively minimal sample and cocktail volumes (Figure 1). The set of 1,536 unique cocktails are based on conditions previously determined to be conducive to protein crystal growth and are designed to be chemically diverse in order to sample a large range of possible crystallization conditions20,21,22. The broad sampling of crystallization conditions increases the likelihood of observing one or more crystallization leads.
Few formal analyses of how many conditions are needed for screening have appeared in the literature. One study focused on the sampling layout of different screens and found that the random sampling of components (similar to an incomplete factorial) represented the most thorough and efficient sampling method23. Another study of screening noted that there have been numerous instances when the very thorough 1,536 screen has yielded only a single crystal hit24, and a very recent study highlighted that most commercial screens undersample the crystallization space known to be associated with screening hits25. Not all crystallization leads will yield a diffraction quality crystal suitable for data collection due to inherent disorder within the crystal, diffraction limitations, or crystal flaws; therefore, casting a wider net for conditions has the additional benefit of providing alternative crystal forms for optimization.
The format of protein crystallization experiments also has an impact on the success of the screen. Vapor diffusion is the most commonly used setup for high-throughput crystallization applications and is utilized at state-of-the art crystallization centers, including the EMBL Hamburg and Institut Pasteur high-throughput screening centers26,27,28. The HTX Center uses the microbatch-under-oil method; while less commonly used, it is a robust method that minimizes the consumption of sample and crystallization cocktails20,21,22. One advantage of the microbatch-under-oil method, particularly when using a high-viscosity paraffin oil, is that only slight evaporation occurs within the drop during the experiment, meaning that the equilibrium concentration is achieved upon drop mixing. If positive crystallization results are observed in the microbatch-under-oil method, the reproduction of these conditions is typically more straightforward than in vapor diffusion setups, in which crystallization occurs at some undefined point during the equilibration between the crystallization drop and the reservoir. The reproducibility of hits is desirable for high-throughput crystallization approaches, which produce prohibitively tiny protein crystals that typically need to be optimized for single-crystal X-ray experiments.
The high-throughput crystallization screen for soluble proteins is made up of cocktails that are prepared in-house, ready-made commercial screens, and in-house-modified commercial screens22. The cocktails were initially developed using the incomplete factorial strategy using previously successful crystallization cocktails20. The reagents in the screen that are commercially available include arrays of polymers, crystallization salts, PEG, and ion combinations and screens that utilize sparse matrix and incomplete factorial approaches. There are also reagents that are modified before inclusion in the screen: an additive screen, a pH and buffer screen, an ionic liquid additive screen, and a polymer screen.
The power of known crystallization conditions and strategies has been leveraged in the 1,536 crystallization cocktails, along with the benefits of the microbatch-under-oil system to generate a pipeline that employs automated liquid handling, automated brightfield imaging, and second order nonlinear imaging of chiral crystals (SONICC). The automation of both the liquid handling and imaging provides the benefits of fewer wet lab hours and higher reproducibility. The high-throughput nature of automated crystallization screening necessitates the automation of the process of monitoring for crystal growth. These advances are achieved with state-of-the-art imaging technologies to assist in the identification of positive crystal hits. Both standard brightfield imaging of plates, as well as multi-photon methods for enhanced detection, are used via a crystal imaging system with SONICC (Figure 2). SONICC combines second harmonic generation (SHG)29 microscopy and ultraviolet two-photon excited fluorescence (UV-TPEF)30 microscopy to detect very small crystals, as well as those obscured by precipitate. The SONICC imaging informs on whether the wells contain protein (via UV-TPEF) and crystals (via SHG). Beyond the positive identification of protein crystals, additional information can also be obtained using state-of-the-art imaging methods. Cocktail-only imaging prior to sample addition serves as a negative control; these images can identify the well appearance prior to sample addition, including in terms of salt crystals and debris. Additionally, SHG and UV-TPEF imaging help differentiate protein crystals from salt crystals and can be used for visualizing protein-nucleic acid complexed material31.
High-throughput crystallization experiments undergoing repeated monitoring via imaging result in a very large volume of images needing examination. Automated crystal scoring methods have been developed to reduce the burden on the user and increase the probability of identifying positive crystal hits. The HTX Center partcipated in the development of the MAchine Recognition of Crystallization Outcomes (MARCO) scoring algorithm, a trained deep convolutional neural network architecture developed by a consortium of academic, non-profit, government, and industry partners to classify brightfield well images32. The algorithm was trained on nearly half a million brightfield images from crystallization experiments from multiple institutions using different crystallization methods and different imagers. The algorithm outputs a probabilistic score indicating whether a given image falls into four possible image classes: &#34;crystal&#34;, &#34;clear&#34;, &#34;precipitate&#34;, and &#34;other&#34;. MARCO has a reported classification accuracy of 94.5%. Crystal detection is further enhanced with software that implements the algorithm and provides a graphical user interface (GUI) for accessible and simple image viewing, enabled with the AI-enabled scoring capabilities32,33. The MARCO Polo GUI is designed to work seamlessly with the setup of the imaging and data management system in the HTX Center to identify hits in the 1,536-well screen, with human engagement to examine the output of sorted lists. Additionally, as open-source software available on GitHub, the GUI is readily available for modification to reflect the specific needs of other laboratory groups.
Here, the process of setting up a high-throughput microbatch-under-oil experiment using robotic liquid handling to deliver both the cocktail and protein is described. The HTX Center has a unique array of instrumentation and resources that are not found at other institutions, with the goal of providing screening services and educational resources to interested users. Demonstrating the methods and capabilities of robotics-enabled high-throughput techniques will enable the community to have knowledge of available technologies and make decisions for their own structure determination efforts.

作者聚焦：高通量筛选以获得蛋白质晶体学的晶体命中  

高通量筛选以获得用于蛋白质晶体学的晶体命中

Getting a crystal to perform diffraction experiments remains a challenge because it's difficult to predict what parameters will influence crystallization. We favor the experimenter by screening 1536 crystallization conditions in a single plate and use advanced imaging technologies to identify even the smallest crystal hits. Structural biology techniques are developing rapidly and the field has been revolutionized by computational structure prediction.This has caused the field to become more integrative with several experimental or computational approaches more commonly being combined to generate a more detailed and accurate representation of biological mechanisms. Generating crystal hits is a key step to performing single crystal x-ray diffraction experiments and to developing techniques like micro ED and serial crystallography. Using advanced imaging methods, UVTBF and SHG along with the power of the Marco algorithm helps us identify useful crystals at all size scales.Our high throughput crystallization methods have generated a large amount of data for probing questions regarding efficiency of crystallization cocktail components. The specialized imaging we do reveal how non-linear optical methods can be used to detect vanishingly small crystals. Finding crystallization conditions is critical for crystal based structural methods, which account for 90%of all structural models in the protein data bank.In 2021, close to 2 million files were downloaded per day from the PDB emphasizing the impact structures have in paving the way for further scientific investigations.

Watch this Scientific Journal Video about Protein Sample Setup and Crystallization at JoVE.com

Protein Sample Setup and Crystallization  

蛋白质样品设置和结晶

将未包装的解冻样品在室温下以10， 000g离心两分钟。观察样品以识别任何沉淀、颜色和条件。将准备好的1， 536孔板加热至23摄氏度，然后以150g离心5分钟。使用明场显微镜拍摄仅用作阴性对照的含有鸡尾酒的平板的照片。使用液体处理机器人将200纳升的样品分配到每个孔中。将板以150g离心后，在所需温度下孵育六周。从第1天到第6周，每周一次捕获带有样品的板的明场图像。使用 SHG 和 UV-TPEF 进行声波成像。在整个实验过程中，自动板成像允许对快速和缓慢生长的晶体进行明场鉴定。UV-TPEF成像表明了蛋白质性质，而SHG成像证实了样品的结晶性质。声波成像能够识别被沉淀或薄膜遮挡的晶体，这些晶体不会产生SHG信号，或者可能被误认为是沉淀物的微晶体。还鉴定出缺乏UV-TPEF信号但显示强明场和SHG信号的非蛋白质晶体。

Research

Education

JoVE Journal

JoVE Core

Analytical Chemistry

Biochemistry

Protein Sample Setup and Crystallization

Unit 1

Introduction to Separation Methods

SCIENTISTS IN ACTION

539 次观看.  Hauptman-Woodward Medical Research Institute. 将未包装的解冻样品在室温下以10， 000g离心两分钟。观察样品以识别任何沉淀、颜色和条件。将准备好的1， 536孔板加热至23摄氏度，然后以150g离心5分钟。使用明场显微镜拍摄仅用作阴性对照的含有鸡尾酒的平板的照片。使用液体处理机器人将200纳升的样品分配到每个孔中。将板以150g离心后，在所需温度下孵育六周。从第1天到第6周，每周一次捕获带有样品的板?...