Determining Macromolecular Structures Using Cryo-Electron Microscopy.

Structural insights into macromolecular and protein complexes provide key clues about the molecular basis of the function. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful structural biology method for studying protein and macromolecular structures at high resolution in both native and near-native states. Despite the ability to get detailed structural insights into the processes underlying protein function using cryo-EM, there has been hesitancy amongst plant biologists to apply the method for biomolecular interaction studies. This is largely evident from the relatively fewer structural depositions of proteins and protein complexes from plant origin in electron microscopy databank. Even though the progress has been slow, cryo-EM has significantly contributed to our understanding of the molecular biology processes underlying photosynthesis, energy transfer in plants, besides viruses infecting plants. This chapter introduces sample preparation for both negative-staining electron microscopy (NSEM) and cryo-EM for plant proteins and macromolecular complexes and data analysis using single particle analysis for beginners.

Preyssler-type phosphotungstate is a new family of negative-staining reagents for the TEM observation of viruses.

Transmission electron microscopy (TEM) is an essential method in virology because it allows for direct visualization of virus morphology at a nanometer scale. Negative staining to coat virions with heavy metal ions must be performed before TEM observations to achieve sufficient contrast. Herein, we report that potassium salts of Preyssler-type phosphotungstates (K(15-n)[P5W30O110Mn+], M = Na+, Ca2+, Ce3+, Eu3+, Bi3+, or Y3+) are high-performance negative staining reagents. Additionally, we compare the staining abilities of these salts to those of uranyl acetate and Keggin-type phosphotungstate. The potassium salt of Preyssler-type phosphotungstates has the advantage of not requiring prior neutralization because it is a neutral compound. Moreover, the potassium counter-cation can be protonated by a reaction with H+-resin, allowing easy exchange of protons with other cations by acid-base reaction. Therefore, the counter-cations can be changed. Encapsulated cations can also be exchanged, and clear TEM images were obtained using Preyssler-type compounds with different encapsulated cations. Preyssler-type phosphotungstates may be superior negative staining reagents for observing virus. Polyoxotungstates (tungsten-oxide molecules with diverse molecular structures and properties) are thus promising tools to develop negative staining reagents for TEM observations.

Double cone-unit laparoscopic hepatic resection using indocyanine green negative counterstaining (with video).

A hepatic cone-unit represents an anatomical unit dominated by a smaller Glissonean pedicle. Anatomical resection of a tumor located in an intersegmental plane is challenging, but could be achieved effectively by performing multiple cone-unit resection. We performed double cone-unit laparoscopic resection of hepatocellular carcinoma located on the intersegmental plane between segments 6a, b. The liver parenchyma covering the posterior Glissonean pedicle was divided along Rouviere's sulcus, the Glissonean branches of segments 6a, b were isolated and ligated, and indocyanine green (ICG) negative counterstaining was performed. The hepatic parenchyma was dissected along the demarcation line to identify the right hepatic vein and the double cone-unit resection was then completed with a negative surgical margin. Thus, double cone-unit laparoscopic hepatectomy with ICG negative counterstaining may be a feasible option for tumors located in an intersegmental plane.

Purification of Human Complement Component C4 and Sample Preparation for Structural Biology Applications.

Activated complement component C4 (C4b) is the nonenzymatic component of the classical pathway (CP) convertases of the complement system. Preparation of C4 and C4b samples suitable for structural biology studies is challenging due to low yields and complexity of recombinant C4 production protocols reported so far and heterogeneity of C4 in native sources. Here we present a purification protocol for human C4 and describe sample preparation methods for structural investigation of C4 and its complexes by crystallography, small angle X-ray scattering, and electron microscopy.

Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples.

In recent years, electron cryo-microscopy (CryoEM) has become a powerful method for the high-resolution studies of biological macromolecules. While CryoEM experiments can begin without additional microscopy steps, negative-stain EM can tremendously minimize CryoEM screening. Negative-stain is a quick method that can be used to screen for robust biochemical conditions, the integrity, binding, and composition of samples and to get an estimation of sample grid concentration. For some applications, the map resolutions potentially afforded by stain may be as biologically informative as in CryoEM. Here, I describe the benefits and pitfalls of negative-stain EM, with particular emphasis on Uranyl stains with the main goal of screening in advance of CryoEM. In addition, I provide a materials list, detailed protocol and possible adjustments for the use of stains for biological samples requiring imaging and/or diffraction-based methods of EM.

LoTToR: An Algorithm for Missing-Wedge Correction of the Low-Tilt Tomographic 3D Reconstruction of a Single-Molecule Structure.

A single-molecule three-dimensional (3D) structure is essential for understanding the thermal vibrations and dynamics as well as the conformational changes during the chemical reaction of macromolecules. Individual-particle electron tomography (IPET) is an approach for obtaining a snap-shot 3D structure of an individual macromolecule particle by aligning the tilt series of electron tomographic (ET) images of a targeted particle through a focused iterative 3D reconstruction method. The method can reduce the influence on the 3D reconstruction from large-scale image distortion and deformation. Due to the mechanical tilt limitation, 3D reconstruction often contains missing-wedge artifacts, presented as elongation and an anisotropic resolution. Here, we report a post-processing method to correct the missing-wedge artifact. This low-tilt tomographic reconstruction (LoTToR) method contains a model-free iteration process under a set of constraints in real and reciprocal spaces. A proof of concept is conducted by using the LoTToR on a phantom, i.e., a simulated 3D reconstruction from a low-tilt series of images, including that within a tilt range of ±15°. The method is validated by using both negative-staining (NS) and cryo-electron tomography (cryo-ET) experimental data. A significantly reduced missing-wedge artifact verifies the capability of LoTToR, suggesting a new tool to support the future study of macromolecular dynamics, fluctuation and chemical activity from the viewpoint of single-molecule 3D structure determination.

Membrane Protein Cryo-EM: Cryo-Grid Optimization and Data Collection with Protein in Detergent.

Cryo-electron microscopy (cryo-EM) is a powerful tool for investigating the structure of macromolecules under near-native conditions. Especially in the context of membrane proteins, this technique has allowed researchers to obtain structural information at a previously unattainable level of detail. Specimen preparation remains the bottleneck of most cryo-EM research projects, with membrane proteins representing particularly challenging targets of investigation due to their universal requirement for detergents or other solubilizing agents. Here we describe preparation of negative staining and cryo-EM grids and downstream data collection of membrane proteins in detergent, by far the most common solubilization agent. This protocol outlines a quick and straightforward procedure for screening and determining the structure of a membrane protein of interest under biologically relevant conditions.

Fast Small-Scale Membrane Protein Purification and Grid Preparation for Single-Particle Electron Microscopy.

The ongoing development of single-particle cryo-electron microscopy (cryo-EM) is leading to fast data acquisition, data processing, and protein structure elucidation. Quick and reliable methods to go from protein purification and optimization to grid preparation will significantly improve the reach and power of cryo-EM. Such methods would particularly constitute a tremendous advantage in structural biology of membrane proteins, whose published structures stay still far behind the number of soluble protein structures. Here we describe a fast, low-cost, and user-friendly method for the purification and cryo-EM analysis of a recombinant membrane protein. This method minimizes the amount of starting material and manipulation steps needed to go from purification to grid preparation, and could potentially be expanded to other membrane protein purification systems for its direct application in structure determination by single-particle cryo-EM.

Optimized Negative-Staining Protocol for Lipid-Protein Interactions Investigated by Electron Microscopy.

A large number of proteins are capable of inserting themselves into lipids, and interacting with membranes, such as transmembrane proteins and apolipoproteins. Insights into the lipid-protein interactions are important in understanding biological processes, and the structure of proteins at the lipid binding stage can help identify their roles and critical functions. Previously, such structural determination was challenging to obtain because the traditional methods, such as X-ray crystallography, are unable to capture the conformational and compositional heterogeneity of protein-lipid complexes. Electron microscopy (EM) is an alternative approach to determining protein structures and visualizing lipid-protein interactions directly, and negative-staining (OpNS), a subset of EM techniques, is a rapid, frequently used qualitative approach. The concern, however, is that current NS protocols often generate artifacts with lipid-related proteins, such as rouleaux formation from lipoproteins. To overcome this artifact formation, Ren and his colleagues have refined early NS protocols, and developed an optimized NS protocol that validated by comparing images of lipoproteins from cryo-electron microscopy (cryo-EM). This optimized NS protocol produces "near native-state" particle images and high contrast images of the protein in its native lipid-binding state, which can be used to create higher-quality three-dimensional (3D) reconstruction by single-particle analysis and electron tomography (e.g. IPET). This optimized protocol is thus a promising hands-on approach for examining the structure of proteins at their lipid-binding status.

Recombinant Expression, Purification, and Assembly of p62 Filaments.

This chapter describes the recombinant overexpression of the canonical selective autophagy receptor p62/SQSTM1 in E. coli and affinity purification. Also described is the method to induce p62 filament assembly and their visualization by negative stain electron microscopy (EM). In cells, p62 forms large structures termed p62 bodies and has been shown to be aggregation prone. This tendency to aggregate poses problems for expression and purification in vitro, which is a prerequisite for structural analysis. Here, we describe the method to express and purify soluble p62, using the solubility tag, MBP, in conjunction with autoinduction. Furthermore, we describe the protocol to assemble p62 into filaments by controlling the ionic strength of its buffer, as well as the preparation of negative stain EM grids to visualize the filaments. In vitro formed p62 filaments can be used to study receptor cargo interactions in minimal reconstituted autophagy model systems.

Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy.

We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody-antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody-antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA. Graphical Abstract ᅟ.

Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems.

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of contrast enhancing stain reagent. Although limited in resolution to a maximum of ~18 - 20 Å, negative stain EM is useful for a variety of biological problems and also provides a rapid means of assessing samples for cryo-electron microscopy (cryo-EM). The negative stain workflow is straightforward method; the sample is adsorbed onto a substrate, then a stain is applied, blotted, and dried to produce a thin layer of electron dense stain in which the particles are embedded. Individual samples can, however, behave in markedly different ways under varying staining conditions. This has led to the development of a large variety of substrate preparation techniques, negative staining reagents, and grid washing and blotting techniques. Determining the most appropriate technique for each individual sample must be done on a case-by-case basis and a microscopist must have access to a variety of different techniques to achieve the highest-quality negative stain results. Detailed protocols for two different substrate preparation methods and three different blotting techniques are provided, and an example of a sample that shows markedly different results depending on the method used is shown. In addition, the preparation of some common negative staining reagents, and two novel Lanthanide-based stains, is described with discussion regarding the use of each.

Analysis of Lipids and Lipid Rafts in Borrelia.

Lipid rafts are membrane microdomains that are involved in cellular processes such as protein trafficking and signaling processes, and which play a fundamental role in membrane fluidity and budding. The lipid composition of the membrane and the biochemical characteristics of the lipids found within rafts define the ability of cells to form microdomains and compartmentalize the membrane. In this chapter, we describe the biophysical, biochemical, and molecular approaches used to define and characterize lipid rafts in the Lyme disease agent, Borrelia burgdorferi.

Negative staining of the vitreous with the use of vital dyes.

PURPOSE: The vitreous cortex, epiretinal membrane (ERM), and inner limiting membrane (ILM) are transparent tissues and are thus difficult to visualize. Staining these structures can increase the efficiency of a nontraumatic removal. METHODS: The surgeon performs a partial core vitrectomy and induces a posterior vitreous detachment. The vital dye is then injected into the retrohyaloid space in balanced salt solution (BSS). The dyes used are TWIN (Alchimia srl, Padova, Italy), MembraneBlue-Dual (DORC International, Zuidland, the Netherlands), and Doubledyne (Alfa Intes, Casoria, Italy). The surgeon can complete the vitrectomy and gradually aspirate the dye with the probe. Once the vitrectomy is complete, the surgeon can perform the peeling of the ERM without the need to reinject the vital dye over the macula. RESULTS: The presence of the dye over the macula facilitates visualization of the vitreous cortex by blocking the red reflex and increasing the contrast power of the coaxial light probe during the vitrectomy. This allows a negative coloration of the vitreous because the dye acts by increasing the visibility of the surrounding BSS and not the vitreous itself. CONCLUSIONS: We describe a new chromovitrectomy technique using the same dye to increase the visualization of the vitreous, posterior hyaloid, ERM, and ILM.

Immunoelectron Microscopy for Visualization of Nanoparticles.

Immunoelectron microscopy (IEM) on a solid phase such as a carbon film is a fast and powerful way to detect and visualize surface antigens on nanoparticles by using a transmission electron microscope (TEM). Nanoparticles, in particular ones for medical applications, are often modified on the surface with soft materials to make them more soluble, less toxic, or targetable to cancerous tumors. Imaging the soft material on the surface of solid nanoparticles by electron microscopy is often a challenge. IEM can overcome this issue in cases where antibodies to any of the surface material are available, which is often the case for proteins, but also for commonly used materials such as polyethylene glycol (PEG). This effective procedure has been used traditionally for viruses and macromolecules, but it can be directly applied to nanoparticles.

Imaging of Liposomes by Transmission Electron Microscopy.

TEM is an important method for the characterization of size and shape of nanoparticles as it can directly visualize single particles and even their inner architecture. Imaging of metal particles in the electron microscope is quite straightforward due to their high density and stable structure, but the structure of soft material nanoparticles, such as liposomes, needs to be preserved for the electron microscope. The best method to visualize liposomes close to their native structure is cryo-electron microscopy, where thin films of suspensions are plunge frozen to create vitrified ice films that can be imaged directly in the electron microscope under liquid nitrogen temperature. Although subject to artifacts, negative staining TEM can also be a useful method to image liposomes, as it is faster and simpler than cryo-EM, and requires less advanced equipment.

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment.

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

Single-Particle Electron Microscopy Analysis of DNA Repair Complexes.

DNA repair complexes play crucial roles in maintaining genome integrity, which is essential for the survival of an organism. The understanding of their modes of action is often obscure due to limited structural knowledge. Structural characterizations of these complexes are often challenging due to a poor protein production yield, a conformational flexibility, and a relatively high molecular mass. Single-particle electron microscopy (EM) has been successfully applied to study some of these complexes as it requires low amount of samples, is not limited by the high molecular mass of a protein or a complex, and can separate heterogeneous assemblies. Recently, near-atomic resolution structures have been obtained with EM owing to the advances in technology and image processing algorithms. In this chapter, we review the EM methodology of obtaining three-dimensional reconstructions of macromolecular complexes and provide a workflow that can be applied to DNA repair complex assemblies.

A method for rapid and sensitive negative staining of proteins in SDS-PAGE using 2',7'-dichlorofluorescein.

We report here a rapid and sensitive technique for negative visualization of protein in 1D and 2D SDS-PAGE by using 2', 7'-dichlorofluorescein (DCF), which appeared as transparent and colorless bands in an opaque gel matrix background. For DCF stain, down to 0.1-0.2 ng protein could be easily visualized within 7 min by only two steps, and the staining is fourfold more sensitive than that of Eosin Y (EY) negative stain and glutaraldehyde (GA) silver stain, and eightfold more sensitive than that of the commonly used imidazole-zinc (IZ) negative stain. Furthermore, DCF stain provided good reproducibility, linearity, and MS compatibility compared with those of IZ stain. In addition, the potential staining mechanism was investigated by colorimetric experiment and molecular docking, and the results demonstrated that the interaction between DCF and protein occurs mainly via van der waals force, electrostatic interaction, and hydrogen bonding.

Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals.

Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures.