introduction
In contemporary biological science research, cryo-electron microscopy (cryo-EM) technology is widely used because of its ability to observe the three-dimensional structure of biological macromolecules at near-atomic resolution. However, interface effects during sample preparation often lead to structural damage to the sample, affecting imaging quality. Although traditional liquid electrospray (ESI) has been applied to sample preparation to reduce these adverse effects, how to optimize ESI parameters to maintain the integrity and functional status of proteins is still an urgent scientific problem to be solved. The study, "Electrospray-assisted cryo-EM sample preparation to mitigate interfacial effects," published April 25 in Nature Methods, mitigates the effects of interface effects by optimizing electrospray-assisted cryo-electron microscopy sample preparation. The research team systematically adjusted key parameters such as sample flow rate, spray voltage, and protein concentration, and conducted a series of experiments using APO-ferritin (apo-ferritin) and angiotensin-converting enzyme 2 (ACE2) as model proteins. The study found that moderate sample flow rates, lower spray voltages, and higher protein concentrations helped to maintain the structural integrity of proteins, and that the use of low-conductivity metallic materials significantly improved the structural retention of protein samples in nano-ESI. These optimization measures significantly improve the imaging quality of the samples and provide effective methodological support for the elucidation of high-resolution protein structures using cryo-EM technology. The results of this study not only improve the application efficiency and accuracy of cryo-EM in protein structure elucidation, but also provide a new perspective for understanding the function of proteins in complex biological processes. The successful implementation of this research is of great significance to promote the development of scientific research and technology in the field of biomedicine, and will contribute to future applications in drug design and disease treatment.
Highlights
This study is the first to elaborate on the process of optimizing protein sample preparation by electrospray-assisted cryo-electron microscopy. By adjusting the sample flow rate, spray voltage, and protein concentration, the original structural and functional state of the protein is effectively maintained, which is difficult to achieve with traditional methods.
The research team conducted systematic optimization experiments to identify the optimal sample flow rate, spray voltage, and protein concentration, which are critical to maintaining the structural integrity of the protein. In addition, the use of low-conductivity metal materials in nano-electrospray (nano-ESI) also significantly improved the structural preservation of samples.
Through optimized parameter settings, the structural integrity of the sample under cryo-EM was significantly improved. This provides a strong methodological support for high-resolution protein structure analysis using cryo-EM technology.
The successful application of this technology not only improves the practicability and efficiency of cryo-EM technology, but also lays a foundation for the wide application of cryo-EM technology in more biological macromolecular structure studies in the future. This will be particularly beneficial in areas such as drug design and disease mechanism research.
Strategies
This study focused on optimizing the electrospray (ESI) parameters during cryo-electron microscopy (cryo-EM) sample preparation to reduce the negative effects of interface effects on protein structural integrity during sample preparation. In this study, parameters such as sample flow rate, spray voltage, protein concentration, and injection distance were adjusted to explore their effects on sample structure preservation. Experimental validation was carried out using well-preserved protein models, such as APO-ferritin (apo-ferritin) and angiotensin-converting enzyme 2 (ACE2), to ensure that the data obtained were universal and reproducible.
Sample preparation studies use purified APO-ferritin and ACE2 as test proteins. These proteins are purified by conventional methods and then diluted to different concentrations and prepared for use in electrospray.
Electrospray Parameters Optimize Sample Flow Rates: Different flow rates from a few microliters to tens of microliters per minute were tested to observe the effects of different flow rates on protein samples. Spray voltage: Adjust the voltage from a few thousand volts to tens of kilovolts and analyze how voltage changes affect the charge state and structural integrity of proteins. Protein concentration: Changes in protein concentration directly affect the aggregation and dispersion behavior of samples during electrospray. Ejection distance: The distance of the sample from the electrical nozzle to the slide, adjusting this parameter to optimize the evaporation and cooling process before the sample lands.
Structural analysis uses cryo-electron microscopy to image the processed samples, collect data, and perform 3D structural reconstruction and analysis using image processing software such as ChimeraX and PHENIX.
Data Statistics and AnalysisA large number of image data obtained in the experiment were statistically analyzed to evaluate the proportion of structural integrity of the samples under different parameters. Negative staining EM and native MS were used to evaluate the retention of samples.
Through detailed parameter adjustments, the study showed that the structure of the sample was best maintained at low flow rates, low voltages, and high protein concentrations. These conditions help to reduce the damage of proteins at the air-liquid interface, thus preserving their original three-dimensional structure in a frozen environment. In addition, the use of low-conductivity metal materials has also shown significant structural protection effects in nano-electrospray (nano-ESI) technology.
Behind the Scenes
Cryo-electron microscopy (cryo-EM) technologyCryo-electron microscopy (cryo-EM) technology is an advanced electron microscopy technique used to observe the near-atom-resolution structure of biological macromolecules. This technique is especially important for biological research, as it allows researchers to observe complex biomolecules, such as proteins and viruses, in a near-natural state.
The basic process of cryo-electron microscopy is sample preparation: the biological sample is first dispersed in droplets on a support grid. It is then frozen rapidly, usually using liquid nitrogen or liquid helium, so that the water molecules form a vitreous state instead of crystals. This flash freezing process, known as "flash freezing", prevents the formation of ice crystals, which is essential for the preservation of the sample structure. Electron microscopy imaging: Frozen samples are placed in an electron microscope at low temperatures (usually liquid nitrogen temperature, about -196 °C). The high-energy electron beam penetrates the sample to form an image. Because the sample remains fixed at very low temperatures, high-resolution imaging can be performed with little to no damage. Image Processing and 3D Reconstruction: Thousands of 2D projected images are collected and processed by sophisticated algorithms to reconstruct the 3D structure of biological macromolecules. This includes aligning, classifying, and averaging images using a variety of computational methods to improve signal-to-noise ratio and resolution. Model construction and validation: After obtaining a high-resolution 3D density map, the atomic model can be fitted in it and further analyzed and validated to ensure the accuracy of the model.
Technological development1975: Introduction of frozen sample preparationJacques Dubochet and colleagues developed the method for frozen water samples, which is the basis of cryo-EM technology. This method avoids the formation of ice crystals by flash-freezing the sample, thus preserving the native state of the biological sample under the electron microscope.
1984: Acquisition of the first cryo-EM imageAlasdair McDowall et al. obtained the first electron microscope image of an ice-free structure using cryo-freezing, marking an important breakthrough in the imaging of biological samples by cryo-EM.
1990s: Development of automated data collection and image processing softwareWith advances in computer technology and image processing algorithms, automated data collection and the development of advanced image reconstruction methods, it became possible to reconstruct complex 3D structures from thousands of 2D images.
2012: The introduction of Direct Electron Detectors significantly improves the signal-to-noise ratio and temporal resolution of images, allowing researchers to obtain clearer and higher-resolution images. This advancement has greatly advanced the application of cryo-EM technology, which enables it to resolve structures close to the atomic level.
2013 and 2015: The Resolution Revolution In these two years, several research groups have reported on the use of cryo-EM to resolve the structure of biological macromolecules at atomic resolution. This is widely considered to be a turning point in the field of cryo-EM, and the technique has since become a powerful tool in structural biology.
2017: The Nobel Prize in Chemistry was jointly awarded to Jacques Dubochet, Joachim Frank and Richard Henderson in Chemistry for their contributions to the development of cryo-EM technology, especially in high-resolution structure determination.
ApplicationsCryo-electron microscopy (cryo-EM) technology is used in a wide range of applications, especially in biology and medicine, and provides a powerful tool for understanding the structure and function of complex biomolecules. Key application areas include:
Protein structure elucidationCryo-EM has a wide range of applications in protein structural biology, especially for large molecules and complexes that are difficult to elucidate by X-ray crystallography or nuclear magnetic resonance (NMR) techniques. It can reveal the three-dimensional structure of proteins, helping researchers understand their function and biochemical properties.
Virology ResearchIn the field of virology, cryo-EM is able to delineate in detail the structure of viral particles, including the arrangement of their surface proteins and genetic material. This is essential to study how viruses infect host cells and how to design effective vaccines and antiviral drugs.
By elucidating the complex structure of a drug molecule with its target, such as a protein or other biological macromolecule, cryo-EM can help scientists design more precise drug molecules to improve drug potency and selectivity.
The biomacromolecular complex Cryo-EM is particularly suitable for the study of large complexes composed of multiple proteins or nucleic acids as well as other molecules. These macromolecular machines often assume key functions in cells, such as ribosomes, membrane protein complexes, molecular motors, etc.
In addition to static structure, cryo-EM is also able to capture the structural changes of biomolecules in different functional states. This has important implications for understanding how biomolecules perform biological functions through their structural changes.
Although nanotechnology and materials science are primarily applied in the field of biology, cryo-EM is also used in nanotechnology and materials science, for example when studying the assembly and structure of nanoparticles and other complex materials.
Difficulties in Sample PreparationSample preparation is a critical and challenging step in cryo-electron microscopy (cryo-EM) because it directly affects the quality and resolution of the final image. Common challenges in sample preparation include:
Sample Concentration and Purity: Samples must be of sufficient purity and appropriate concentration. Impurities and contaminants can interfere with image quality, and concentrations that are too high or too low can affect the distribution of the sample on the support grid and the final data quality.
Freezing technologyFreezing is at the heart of cryo-EM and requires the sample to be quickly frozen below the temperature of liquid nitrogen to avoid the formation of ice crystals. The presence of ice crystals can interfere with the electron beam and affect the resolution of the image. Achieving ideal freezing conditions requires precise control of the temperature and freezing rate of the sample.
Uniform distribution of the sample on the support membrane The sample needs to be evenly distributed on the support grid and of moderate thickness. Inhomogeneous or clustered samples can lead to inconsistent data collection, which can affect the quality of the final 3D reconstruction.
Dehydration ProtectionDuring freezing, it is ideal for water molecules to solidify into an amorphous "glassy state" without the formation of ice crystals. This requires the transition of the sample from liquid to solid state in a very short time, and any operational defects can lead to sample damage or structural distortion.
The quality and handling of EM grids supported by EM grids and the way they are handled are also critical to sample preparation. The grids need to be properly cleaned and handled (e.g., with colloidal films) to ensure that the sample is evenly distributed and adhesive.
Stability and sensitivity of the sampleBiological samples may be sensitive to environmental conditions, including temperature, pH, and exposure to the electron beam. This requires tightly controlled experimental conditions during sample preparation and EM operation.
Repeatability and reliability to ensure consistent quality during repetitive sample preparation is a challenge, especially for biomacromolecules with complex structures.
Technology and experience requirements: High-quality sample preparation requires great skill and experience. Laboratory conditions, equipment, and operator skills can all have a significant impact on the results of sample preparation.
Liquid Electrospray Ionization (ESI)The application of Liquid Electrospray Ionization (ESI) technology in cryo-electron microscopy (cryo-EM) mainly involves a specific sample preparation method called electrospray freezing. This method combines traditional electrospray techniques with cryo-electron microscopy to improve the efficiency of sample preparation and the quality of samples.
Electrospray ionization (ESI) was originally a sample ionization technique for mass spectrometry (MS). It works by applying a high voltage to a liquid sample, forming charged droplets, which then produce charged molecules or clusters of molecules through the evaporation of the solvent. This process enables large molecules (e.g., proteins, nucleic acids, etc.) to be mass-analyzed in the gas phase.
Application of Electrospray Freezing in Cryo-EM In cryo-EM, electrospray technology is used to prepare biological samples. Using electrospray freezing, a liquid sample can be quickly frozen as it is ejected through an electrically charged nozzle to form tiny droplets. In this process, sample droplets are sprayed directly into liquid nitrogen or other coolant, which freezes instantaneously and forms amorphous ice, which helps preserve the original state and structure of the sample.
Advantages: Rapid sample processing: Electrospray can quickly generate a large number of sample droplets, making it suitable for high-throughput sample preparation. Minimize sample volume: This technique can be prepared with very small amounts of sample, making it ideal for precious or hard-to-obtain samples. Reduced sample aggregation: With electrospray, the sample is already a fine droplet before freezing, which helps to reduce the aggregation of the sample during freezing.
Challenging equipment and operational complexity: Electrospray freezing requires special equipment and delicate handling techniques. Stability and uniformity of the sample: Ensuring consistency in the size and concentration of the droplets generated by the spray is a challenge. Freezing speed and efficiency: It is important to ensure that sample droplets are frozen quickly and efficiently to avoid the formation of ice crystals.
Charged water droplets during electrosprayTraditionally, it has been suggested that the charged water droplets generated during the final Coulomb fission event will be slightly larger than the macromolecule, and that all the droplet charges will be transferred to the surface of the macromolecule during the final solvent evaporation. However, the study proposed that in their experimental setup, the droplets containing macromolecules did not completely evaporate into the gas phase, so most of these molecules remained submerged in the liquid, with a low degree of protonation compared to the gaseous ionic state.
Relationship between Coulomb Rupture of Charged Water Droplets and MacromoleculesIn the process of electrospray, a liquid sample passes through a nozzle under the action of high voltage to form charged tiny water droplets. As these water droplets fly through the air, the accumulation of surface charge to a certain extent exceeds the surface tension, causing the coulomb to crack and form smaller droplets. Conventional wisdom has been that the solvent in these smaller droplets evaporates on the way to the detector, eventually leaving only charged macromolecules. In this process, all drop charges are thought to be transferred to the surface of the macromolecule, which may alter the spatial structure of the macromolecule, as the charge redistribution affects its three-dimensional configuration.
This discovery has important implications for improving electrospray mass spectrometry techniques. By controlling the degree of evaporation and protonation of water droplets, the native state of the sample can be maintained more precisely, thus improving the accuracy and reliability of structural analysis. In addition, it also provides new ideas for solving the problems that traditional ESI-MS may encounter when processing large molecule samples, such as how to reduce structural perturbations during sample processing.
In the ESI process, the sample solution is ejected through a charged nozzle, forming tiny charged droplets. During the flight to the detector, the droplets undergo solvent evaporation, causing the solutes in the drops, such as protein macromolecules, to gradually concentrate and eventually become charged. In this process, the degree of protonation of a protein – that is, the number of protons on the surface of a protein molecule – will directly affect the preservation of its three-dimensional structure. Protonation is often associated with the structural stability and functional activity of protein molecules. Excessive protonation may lead to deformation of the molecular structure of proteins, which can affect their biological activity. Therefore, how to control the degree of protonation during ESI to maintain the native state of the protein is the key to improving the quality of the analysis. In this study, various parameters of ESI (such as nozzle voltage, flow rate, evaporation rate of droplets, etc.) were controlled to achieve fine control of the degree of protonation. In particular, the experimental results show that large molecules such as proteins can maintain the integrity of their native structure even in extreme environments such as high electric fields and low voltage environments under low protonation conditions. This provides strong experimental support for high-resolution structural analysis of proteins and other macromolecules using ESI technology. This study not only optimizes the sample preparation and processing process in ESI technology, but also provides a new methodology for high-resolution structural analysis of biological macromolecules. By maintaining the high-resolution native structure of macromolecules, the application prospects of ESI technology will be further extended to many fields such as structural biology, drug design, and protein engineering.
Electrochemical Reaction Models: A hypothetical model is proposed in this study to explain electrochemical reactions under different conditions and their effects on protein structure. The study discusses the electrostatic forces and the conversion of kinetic energy into surface potential energy to generate ice layers and their thickness gradients, which are advantageous for accommodating protein complexes of different sizes. In the ESI process, a liquid sample is ejected into tiny droplets of charge under the action of a high voltage. Rapid solvent evaporation occurs during the droplets' flight to the mass spectrometer detector, resulting in a gradual increase in the concentration of solutes (e.g., proteins) inside the droplets. In this process, the charge inside the droplet is redistributed and a strong electrostatic force is generated. These forces can cause structural changes within or on the surface of a protein molecule, which can affect its final detection and analysis results. In addition, the electrochemical reaction model mentioned in the study also takes into account the conversion of kinetic energy to surface potential energy. This energy conversion is able to form a layer of ice with a specific thickness gradient on the surface of the droplets. Ice formation not only helps to reduce the direct exposure of protein molecule surfaces, but also provides a relatively stable microenvironment that helps maintain the structural integrity of protein complexes.
The application of the model and its effect on protein structure The electrochemical reaction model provides a method to optimize ice formation and thickness gradients by manipulating the electric field strength and spray parameters (e.g., flow rate, voltage, etc.) during electrospraying. This method is particularly suitable for working with large molecules with complex structures or susceptibility to environmental influences, such as protein complexes. With this model, the spatial arrangement and structural stability of proteins during sample preparation can be more precisely controlled. For example, by adjusting electrospray parameters to optimize ice formation, protein complexes can be effectively "fixed" in specific spatial configurations, resulting in more accurate and realistic structural information in mass spectrometry.
The application of this electrochemical reaction model not only enhances our understanding of charge dynamics and energy conversion mechanisms in ESI technology, but also provides a new technical means for high-resolution structural analysis of macromolecules such as proteins. This is of great scientific and practical significance for more in-depth research on the structure and function of protein complexes in drug development, biotechnology and related fields in the future.
The improved angular projection coverage of the Euler angle distribution of the five macromolecular samples shows that the angular projection coverage is significantly improved in the Fourier space, which is essential for high-resolution 3D reconstruction.
Euler angles are distributed in cryo-electron microscopy techniques, and are the three angles that describe the orientation of a sample in three-dimensional space—pitch, yaw, and roll. In the 3D reconstruction of macromolecule samples, the 2D projection images in different directions will be synthesized to reconstruct the complete 3D structure. The distribution of Euler angles determines the directional diversity and comprehensiveness of these projections, which in turn directly affects the resolution and accuracy of the final 3D structure.
Improvement of angular projection coverageIn traditional cryo-EM sampling, due to the random arrangement and fixation of samples on the slide, data may be missing or under-covered in some directions, which is called preferential orientation. This preference leads to blind spots in 3D reconstruction, which reduces the resolution and reliability of the structure. By optimizing the electrospray process and sample preparation parameters, the angular projection coverage of the sample can be significantly improved. Specifically, the distribution and orientation of the sample on the slide can be controlled by adjusting the speed and angle of the sample ejection, and the rate of solvent evaporation.
The scientific significance of high-resolution 3D reconstruction By improving the distribution of Euler angles, researchers are able to obtain a more uniform and comprehensive data set, which means that in Fourier space, data from all angles can be fully sampled. This comprehensive data acquisition makes it possible to reconstruct unambiguous and high-precision 3D structures, especially when analyzing the dynamic structure and interactions of biological macromolecules such as protein complexes.
By using advanced electrospray techniques and optimized cryo-EM sample preparation, researchers are able to effectively control the degree of protonation and charge distribution of the sample, which affects its orientation and distribution on the slide. In addition, the use of advanced image processing algorithms and data analysis techniques, such as the use of multivariate statistical analysis and machine learning methods to optimize the angular distribution and image reconstruction process, is also the key to achieving high-resolution 3D structure reconstruction. Therefore, by improving the distribution of Euler angles and the coverage of angular projection, it can not only improve the structural resolution ability of cryo-EM technology, but also promote a deeper understanding of complex biomolecular systems, which has great scientific and application value for biomedical research and related fields.
Potential LimitationsChallenges of Nano-ESI Nano-Electrospray TechnologyIn the implementation of nano-electrospray-assisted cryo-electron microscopy (cryo-EM) sample preparation, it has been pointed out that nanoelectrospray (nano-ESI) faces the problems of particle separation and potential thin film evaporation due to electrochemical effects. These issues can affect the quality and integrity of the sample, limiting the application of the technique in some cases.
Complexity of High-Resolution 3D Structure ReconstructionAlthough electrospray-assisted cryo-EM technology can improve sample processing and 3D structure reconstruction, obtaining accurate data from high-resolution microscopic images remains a technical challenge. Especially when working with ultra-thin samples, maintaining a uniform thickness of the sample and avoiding structural deformation requires precise operation and optimized equipment configuration.
Although the ESI-cryoPrep method is expected to be used in multiple laboratories and research institutions due to its low cost and relatively simple maintenance, its general applicability and efficacy in different types of protein samples still need to be further validated. The applicability of specific protein samples, such as membrane proteins and tiny crystals, has not been fully explored.
The need to optimize technical parametersWhile sample preparation can be optimized by adjusting the ejection conditions, it remains a challenge to precisely control these parameters to suit different types of biomolecules. This includes, but is not limited to, solution injection flow rate, spray voltage, sample concentration, and landing distance from the tip of the nozzle to the grid, which are all variables that need to be fine-tuned in an experiment.
Long-term experimental validation and improvement studies have shown that although the use of electrospray-assisted cryo-EM sample preparation techniques has brought some initial success, the long-term efficacy and reliability of these methods and techniques need to be further validated and improved through more experiments and data analysis.
Potential Research DirectionsOptimizing Nano-ESI Parameters Studies have shown that there are some operational challenges in nanoelectrospray technology, such as particle separation due to electrochemical effects and potential evaporation of thin liquid films. Future research can focus on how to optimize the operating conditions of nanoelectrospray, including voltage, flow rate, solvent selection, etc., to reduce these negative effects. By fine-tuning these parameters, it may be helpful to improve the structural retention of the sample in cryo-EM analysis.
Expanding to different types of biological macromolecule samples, current research has focused on a few model proteins. Future research can apply the method to a wider range of biological macromolecules, such as membrane proteins and tiny crystals. Due to their unique biological functions and structural properties, these biomacromolecules have more stringent requirements for sample preparation and structural analysis. Exploring electrospray-assisted cryo-EM technology for these complex samples will be an important research direction.
Further development of high-resolution 3D structure reconstruction technologyAlthough the existing technology can support high-resolution structural reconstruction, how to further improve the resolution and reconstruction efficiency is still an important research field. Future research could explore the combination of artificial intelligence and machine learning technologies to automate and optimize data processing processes, improve resolution and reduce the time cost of data processing.
An in-depth understanding of the microscopic physical and chemical mechanisms involved in electrospray processes can help researchers better control experimental conditions and optimize sample preparation by studying the complex physical and chemical changes involved in electrospray processes. Future research should focus on the mechanisms underlying droplet formation, charge distribution, solvent evaporation, and how these factors affect the quality and resolution of the final cryo-EM image.
Link to original article
Yang Z, Fan J, Wang J, Fan X, Ouyang Z, Wang HW, Zhou X. Electrospray-assisted cryo-EM sample preparation to mitigate interfacial effects. Nat Methods. 2024 Apr 25. doi: 10.1038/s41592-024-02247-0. Epub ahead of print. PMID: 38664529.
https://www.nature.com/articles/s41592-024-02247-0
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