cryo-EM Grid Research Articles

A cryo-EM grid preparation device for time-resolved structural studies.

Structural biology generally provides static snapshots of protein conformations that can provide information on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualize, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. X-ray free-electron-laser technology has provided a powerful tool to study enzyme mechanisms at atomic resolution, typically in the femtosecond to picosecond timeframe. Complementary to this, recent advances in the resolution obtainable by electron microscopy and the broad range of samples that can be studied make it ideally suited to time-resolved approaches in the microsecond to millisecond timeframe to study large loop and domain motions in biomolecules. Here we describe a cryo-EM grid preparation device that permits rapid mixing, voltage-assisted spraying and vitrification of samples. It is shown that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub-4 Å reconstruction. Rapid mixing can be achieved by blot-and-spray or mix-and-spray approaches with a delay of ∼10 ms, providing greater temporal resolution than previously reported mix-and-spray approaches.

AgarFix: Simple and accessible stabilization of challenging single-particle cryo-EM specimens through crosslinking in a matrix of agar

Cryogenic electron microscopy (cryo-EM) allows structure determination of macromolecular assemblies that have resisted other structural biology approaches because of their size and heterogeneity. These challenging multi-protein targets are typically susceptible to dissociation and/or denaturation upon cryo-EM grid preparation, and often require crosslinking prior to freezing. Several approaches for gentle on-column or in-tube crosslinking have been developed. On-column crosslinking is not widely applicable because of the poor separation properties of gel filtration techniques. In-tube crosslinking frequently causes sample aggregation and/or precipitation. Gradient-based crosslinking through the GraFix method is more robust, but very time-consuming and necessitates specialised expensive equipment. Furthermore, removal of the glycerol typically involves significant sample loss and may cause destabilization detrimental to the sample quality. Here, we introduce an alternative procedure: AgarFix (Agarose Fixation). The sample is embedded in an agarose matrix that keeps the molecules separated, thus preventing formation of aggregates upon cross-inking. Gentle crosslinking is accomplished by diffusion of the cross-linker into the agarose drop. The sample is recovered by diffusion or electroelution and can readily be used for cryo-EM specimen preparation. AgarFix requires minimal equipment and basic lab experience, making it widely accessible to the cryo-EM community.

3.1 Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids

Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact with the air-water interface, which often leads to detrimental effects such as denaturation or adoption of preferred orientations, ultimately hindering structure determination. Randomly biotinylating the protein of interest (for example, at its primary amines) and then tethering it to a cryo-EM grid coated with two-dimensional crystals of streptavidin (acting as an affinity surface) can prevent the protein from interacting with the air-water interface. Recently, this approach was successfully used to solve a high-resolution structure of a test sample, a bacterial ribosome. However, whether this method can be used for samples where interaction with the air-water interface has been shown to be problematic remains to be determined. Here we report a 3.1 Å structure of an RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to solve this structure using conventional sample preparation methods resulted in a poor quality cryo-EM map due to Pol II EC(CPD)’s adopting a strong preferred orientation. Imaging the same sample on streptavidin grids improved the angular distribution of its view, resulting in a high-resolution structure. This work shows that streptavidin affinity grids can be used to address known challenges posed by the interaction with the air-water interface.

Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy.

Correlated light and electron microscopy (CLEM) has become a popular technique for combining the protein-specific labeling of fluorescence with electron microscopy, both at room and cryogenic temperatures. Fluorescence applications at cryo-temperatures have typically been limited to localization of tagged protein oligomers due to known issues of extended triplet state duration, spectral shifts, and reduced photon capture through cryo-CLEM objectives. Here, we consider fluorophore characteristics and behaviors that could enable more extended applications. We describe how dialkylcarbocanine DiD, and its autoquenching by resonant energy transfer (RET), can be used to distinguish the fusion state of a lipid bilayer at cryo-temperatures. By adapting an established fusion assay to work under cryo-CLEM conditions, we identified areas of fusion between influenza virus-like particles and fluorescently labeled lipid vesicles on a cryo-EM grid. This result demonstrates that cryo-CLEM can be used to localize functions in addition to tagged proteins, and that fluorescence autoquenching by RET can be incorporated successfully into cryo-CLEM approaches. In the case of membrane fusion applications, this method provides both an orthogonal confirmation of functional state independent of the morphological description from cryo-EM and a way to bridge room-temperature kinetic assays and the cryo-EM images.

Mammalian Respiratory Complex I Through the Lens of Cryo-EM.

Single-particle electron cryomicroscopy (cryo-EM) has led to a revolution in structural work on mammalian respiratory complex I. Complex I (mitochondrial NADH:ubiquinone oxidoreductase), a membrane-bound redox-driven proton pump, is one of the largest and most complicated enzymes in the mammalian cell. Rapid progress, following the first 5-Å resolution data on bovine complex I in 2014, has led to a model for mouse complex I at 3.3-Å resolution that contains 96% of the 8,518 residues and to the identification of different particle classes, some of which are assigned to biochemically defined states. Factors that helped improve resolution, including improvements to biochemistry, cryo-EM grid preparation, data collection strategy, and image processing, are discussed. Together with recent structural data from an ancient relative, membrane-bound hydrogenase, cryo-EM on mammalian complex I has provided new insights into the proton-pumping machinery and a foundation for understanding the enzyme's catalytic mechanism.

Bioactive Functionalized Monolayer Graphene for High-Resolution Cryo-Electron Microscopy.

Single-particle cryo-electron microscopy (cryo-EM) has become one of the most essential tools to understand biological mechanisms at molecular level. A major bottleneck in cryo-EM technique is the preparation of good specimens that embed biological macromolecules in a thin layer of vitreous ice. In the canonical cryo-EM specimen preparation method, biological macromolecules tend to be adsorbed to the air-water interface, causing partial denaturation and/or preferential orientations. In this work, we have designed and produced a new type of cryo-EM grids using bioactive-ligand functionalized single-crystalline monolayer graphene membranes as supporting films. The functionalized graphene membrane (FGM) grids exhibit specific binding affinity to histidine (His)-tagged proteins and complexes. In cryo-EM, the FGM grids generate relatively low background for imaging and selectively anchor 20S proteasomes to the supporting film surface, enabling near-atomic-resolution 3D reconstruction of the complex. We envision that the FGM grids could benefit single particle cryo-EM specimen preparation with high reproducibility and robustness, therefore enhancing the efficiency and throughput of high-resolution cryo-EM structural determination.

Cryoet of Single Particle CryoEM Grids Reveals Widespread, but Reducible, Particle Adsorption to the Air-Water Interface

Cryoet of Single Particle CryoEM Grids Reveals Widespread, but Reducible, Particle Adsorption to the Air-Water Interface

Characterizing Functional States of a Model Ligand-Gated Ion Channel by Cryo-Electron Microscopy

Pentameric ligand-gated ion channels are important mediators of electrochemical signal transduction in the brain and other systems. Recent progress in protein biochemistry and structural biology has shed light on topology and possible gating mechanisms in these channels, yet the functionally relevant endpoints of activation and modulation remain unclear. In order to obtain alternative structural insights, we optimized protein purification, reconstitution, and grid preparation conditions for structure determination of a prokaryotic ion channel using cryo-electron microscopy (EM). We prepared samples at various presumed distributions of active and inactive states, with the goal of quantifying distinct classes within individual cryo-EM grids. Modification of carbon support, glow-discharging vapor, and blotting time were critical to improving particle distribution and orientation, and molecular simulations were harnessed to validate structural models. This work promises to inform methods development in cryo-EM of membrane proteins, and to populate the gating landscape in this important ion channel family.

Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO

Preferred particle orientation presents a major challenge for many single particle cryo-electron microscopy (cryo-EM) samples. Orientation bias limits the angular information used to generate three-dimensional maps and thus affects the reliability and interpretability of the structural models. The primary cause of preferred orientation is presumed to be due to adsorption of the particles at the air/water interface during cryo-EM grid preparation. To ameliorate this problem, detergents are often added to cryo-EM samples to alter the properties of the air/water interface. We have found that many bacterial transcription complexes suffer severe orientation bias when examined by cryo-EM. The addition of non-ionic detergents, such as NP-40, does not remove the orientation bias but the Zwitter-ionic detergent CHAPSO significantly broadens the particle orientation distributions, yielding isotropically uniform maps. We used cryo-electron tomography to examine the particle distribution within the ice layer of cryo-EM grid preparations of Escherichia coli 6S RNA/RNA polymerase holoenzyme particles. In the absence of CHAPSO, essentially all of the particles are located at the ice surfaces. CHAPSO at the critical micelle concentration coats the air/water interface and eliminates particle absorption there, allowing particles to randomly orient within the vitreous ice layer. We find that CHAPSO reduces orientation bias for a number of bacterial transcription complexes containing E. coli or Mycobacterium tuberculosis RNA polymerases. Findings of this study confirm the presumed basis for how detergents can help remove orientation bias in cryo-EM samples and establishes CHAPSO as a useful tool to facilitate cryo-EM studies of bacterial transcription complexes.

CryoET of Single Particle CryoEM Grids Reveals Widespread Particle Adsorption to the Air-Water Interface, Which May be Reduced with New Plunging Techniques

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Cryo-EM Grid Preparation of Membrane Protein Samples for Single Particle Analysis.

Recent advances in cryo-electron microscopy (cryo-EM) have made it possible to solve structures of biological macromolecules at near atomic resolution. Development of more stable microscopes, improved direct electron detectors and faster software for image processing has enabled structural solution of not only large macromolecular (megadalton range) complexes but also small (~60 kDa) proteins. As a result of the widespread use of the technique, we have also witnessed new developments of techniques for cryo-EM grid preparation of membrane protein samples. This includes new types of solubilization strategies that better stabilize these protein complexes and the development of new grid supports with proven efficacy in reducing the motion of the molecules during electron beam exposure. Here, we discuss the practicalities and recent challenges of membrane protein sample preparation and vitrification, as well as grid support and foil treatment in the context of the structure determination of protein complexes by single particle cryo-EM.

A simple and robust procedure for preparing graphene-oxide cryo-EM grids.

Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited their widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids can be used for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and provide partial protection from the air-water interface. Additionally, the signal of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.

Routine determination of ice thickness for cryo-EM grids.

Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.

Routine single particle CryoEM sample and grid characterization by tomography.

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

Approaches to altering particle distributions in cryo-electron microscopy sample preparation.

Cryo-electron microscopy (cryo-EM) can now be used to determine high-resolution structural information on a diverse range of biological specimens. Recent advances have been driven primarily by developments in microscopes and detectors, and through advances in image-processing software. However, for many single-particle cryo-EM projects, major bottlenecks currently remain at the sample-preparation stage; obtaining cryo-EM grids of sufficient quality for high-resolution single-particle analysis can require the careful optimization of many variables. Common hurdles to overcome include problems associated with the sample itself (buffer components, labile complexes), sample distribution (obtaining the correct concentration, affinity for the support film), preferred orientation, and poor reproducibility of the grid-making process within and between batches. This review outlines a number of methodologies used within the electron-microscopy community to address these challenges, providing a range of approaches which may aid in obtaining optimal grids for high-resolution data collection.

Delivery of femtolitre droplets using surface acoustic wave based atomisation for cryo-EM grid preparation.

Cryo-Electron Microscopy (cryo-EM) has become an invaluable tool for structural biology. Over the past decade, the advent of direct electron detectors and automated data acquisition has established cryo-EM as a central method in structural biology. However, challenges remain in the reliable and efficient preparation of samples in a manner which is compatible with high time resolution. The delivery of sample onto the grid is recognized as a critical step in the workflow as it is a source of variability and loss of material due to the blotting which is usually required. Here, we present a method for sample delivery and plunge freezing based on the use of Surface Acoustic Waves to deploy 6-8 µm droplets to the EM grid. This method minimises the sample dead volume and ensures vitrification within 52.6 ms from the moment the sample leaves the microfluidics chip. We demonstrate a working protocol to minimize the atomised volume and apply it to plunge freeze three different samples and provide proof that no damage occurs due to the interaction between the sample and the acoustic waves.

Methods for Preparing Cryo-EM Grids of Large Macromolecular Complexes.

The recent resolution revolution in cryo-electron microscopy has generated a huge interest in the technique for determining atomic resolution structures of large and dynamic macromolecular complexes that are intractable to crystallography and NMR. A key element of success in cryo-EM is the quality of the specimen vitrified on the cryo-EM grid. In this chapter we outline methods for cryo-EM grid sample preparation.

Design of a High Capacity Puck Storage System for Cryo-EM grids in a Facility Setting

Design of a High Capacity Puck Storage System for Cryo-EM grids in a Facility Setting

A novel storage system for cryoEM samples.

We present here a new CryoEM grid boxes storage system designed to simplify sample labeling, tracking and retrieval. The system is based on the crystal pucks widely used by the X-ray crystallographic community for storage and shipping of crystals. This system is suitable for any cryoEM laboratory, but especially for large facilities that will need accurate tracking of large numbers of samples coming from different sources.

A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM

We describe a spraying-plunging method for preparing cryoelectron microscopy (cryo-EM) grids with vitreous ice of controllable, highly consistent thickness using a microfluidic device. The new polydimethylsiloxane (PDMS)-based sprayer was tested with apoferritin. We demonstrate that the structure can be solved to high resolution with this method of sample preparation. Besides replacing the conventional pipetting-blotting-plunging method, one of many potential applications of the new sprayer is in time-resolved cryo-EM, as part of a PDMS-based microfluidic reaction channel to study short-lived intermediates on the timescale of 10-1,000ms.