High-resolution Single-particle cryo-EM Research Articles

Metallo-supramolecular branched polymer protects particles from air-water interface in single-particle cryo-electron microscopy

Recent technological breakthroughs in single-particle cryo-electron microscopy (cryo-EM) enable rapid atomic structure determination of biological macromolecules. A major bottleneck in the current single particle cryo-EM pipeline is the preparation of good quality frozen cryo-EM grids, which is mostly a trial-and-error process. Among many issues, preferred particle orientation and sample damage by air–water interface (AWI) are common practical problems. Here we report a method of applying metallo-supramolecular branched polymer (MSBP) in the cryo-sample preparation for high-resolution single-particle cryo-EM. Our data shows that MSBP keeps a majority of particles away from air–water interface and mitigates preferred orientation as verified by the analyses of apoferritin, hemagglutinin) trimer and various sample proteins. The use of MSBP is a simple method to improve particle distribution for high-resolution structure determination in single-particle cryo-EM.

High-Resolution Single-Particle Cryo-EM Hydrated Structure of Streptococcus pyogenes Enolase Offers Insights into Its Function as a Plasminogen Receptor.

Cellular plasminogen (Pg) receptors (PgRs) are utilized to recruit Pg; stimulate its activation to the serine protease, plasmin (Pm); and sterically protect the surface Pm from inactivation by host inhibitors. One such PgR is the moonlighting enzyme, enolase, some of which leaves the cytoplasm and resides at the cell surface to potentially function as a PgR. Since microbes employ conscription of host Pg by PgRs as one virulence mechanism, we explored the structural basis of the ability of Streptococcus pyogenes enolase (Sen) to function in this manner. Employing single-particle cryo-electron microscopy (cryo-EM), recombinant Sen from S. pyogenes was modeled at 2.6 Å as a stable symmetrical doughnut-shaped homooctamer with point group 422 (D4) symmetry, with a monomeric subunit molecular weight of ∼49 kDa. Binding sites for hPg were reported in other studies to include an internal K252,255 and the COOH-terminal K434,435 residues of Sen. However, in native Sen, the latter are buried within the minor interfaces of the octamer and do not function as a Pg-binding epitope. Whereas Sen and hPg do not interact in solution, when Sen is bound to a surface, hPg interacts with Sen independently of K252,255,434,435. PgRs devoid of COOH-terminal lysine utilize lysine isosteres comprising a basic residue, "i", and an anionic residue at "i + 3" around one turn of an α-helix. We highlight a number of surface-exposed potential hPg-binding lysine isosteres and further conclude that while the octameric structure of Sen is critical for hPg binding, disruption of this octamer without dissociation exposes hPg-binding epitopes.

PH-tunable membrane-active polymers, NCMNP2a-x, and their potential membrane protein applications.

Accurate 3D structures of membrane proteins are essential for comprehending their mechanisms of action and designing specific ligands to modulate their activities. However, these structures are still uncommon due to the involvement of detergents in the sample preparation. Recently, membrane-active polymers have emerged as an alternative to detergents, but their incompatibility with low pH and divalent cations has hindered their efficacy. Herein, we describe the design, synthesis, characterization, and application of a new class of pH-tunable membrane-active polymers, NCMNP2a-x. The results demonstrated that NCMNP2a-x could be used for high-resolution single-particle cryo-EM structural analysis of AcrB in various pH conditions and can effectively solubilize BcTSPO with the function preserved. Molecular dynamic simulation is consistent with experimental data that shed great insights into the working mechanism of this class of polymers. These results demonstrated that NCMNP2a-x might have broad applications in membrane protein research.

IceBreaker: Software for high-resolution single-particle cryo-EM with non-uniform ice

SummaryDespite the abundance of available software tools, optimal particle selection is still a vital issue in single-particle cryoelectron microscopy (cryo-EM). Regardless of the method used, most pickers struggle when ice thickness varies on a micrograph. IceBreaker allows users to estimate the relative ice gradient and flatten it by equalizing the local contrast. It allows the differentiation of particles from the background and improves overall particle picking performance. Furthermore, we introduce an additional parameter corresponding to local ice thickness for each particle. Particles with a defined ice thickness can be grouped and filtered based on this parameter during processing. These functionalities are especially valuable for on-the-fly processing to automatically pick as many particles as possible from each micrograph and to select optimal regions for data collection. Finally, estimated ice gradient distributions can be stored separately and used to inspect the quality of prepared samples.

High-resolution single-particle cryo-EM of samples vitrified in boiling nitro-gen.

Based on work by Dubochet and others in the 1980s and 1990s, samples for single-particle cryo-electron microscopy (cryo-EM) have been vitrified using ethane, propane or ethane/propane mixtures. These liquid cryogens have a large difference between their melting and boiling temperatures and so can absorb substantial heat without formation of an insulating vapor layer adjacent to a cooling sample. However, ethane and propane are flammable, they must be liquified in liquid nitro-gen immediately before cryo-EM sample preparation, and cryocooled samples must be transferred to liquid nitro-gen for storage, complicating workflows and increasing the chance of sample damage during handling. Experiments over the last 15 years have shown that cooling rates required to vitrify pure water are only ∼250 000 K s-1, at the low end of earlier estimates, and that the dominant factor that has limited cooling rates of small samples in liquid nitro-gen is sample precooling in cold gas present above the liquid cryogen surface, not the Leidenfrost effect. Using an automated cryocooling instrument developed for cryocrystallography that combines high plunge speeds with efficient removal of cold gas, we show that single-particle cryo-EM samples on commercial grids can be routinely vitrified using only boiling nitro-gen and obtain apoferritin datasets and refined structures with 2.65 Å resolution. The use of liquid nitro-gen as the primary coolant may allow manual and automated workflows to be simplified and may reduce sample stresses that contribute to beam-induced motion.

Abstract P-20: Sub 3 Resolution Cryo-EM Structure of Eukaryotic Small (40S) Ribosomal Subunit

Background: The ribosome is a molecular machine that translates mRNAs into proteins. In eukaryotes, ribosome consists of small (40S) and large (60S) subunits. Translation in eukaryotes is a complicated molecular process that involves the formation of various molecular complexes consisting of ribosomal subunits and protein factors. Cryo-EM approaches such as single particle analysis are widely used for structural analysis of components and intermediates of the translation machinery. However, the process of translation in plants is still poorly characterized at a structural level. Here, we present the structure of Triticum aestium small ribosomal subunit obtained at sub 3 resolution that can be used for further structural studies of the translation process in plants. Methods: The structures of the 40S subunits purified from wheat germ extract were obtained using high-resolution single particle cryo-EM. For cryo-EM sample preparation were used Quantifoil R 1.2/1.3 grids coated with an additional 2 nm amorphous carbon film were glow-discharged for 30 seconds at 15 mA using PELCO easiGlow (Ted Pella). 3 µL of the sample were applied onto the grids, blotted for 3 sec at 10°C and 100% humidity, and plunge-frozen in liquid ethane using Vitrobot Mark IV (Thermo Fisher). Cryo-EM data were collected using a Cs-corrected Titan Krios (Thermo Fisher) transmission electron microscope, equipped with a Falcon II direct electron detector. Data were acquired with defocus range of -0.6 to -2.0 at a nominal magnification of 75,000x, giving a calibrated pixel size of 0.86 Å/pixel. The micrographs were recorded as movie stacks. The exposure time for each stack was 1.6 s, corresponding to a total electron dose of ~84 e-/Å2 fractionated into 32 frames (~2.6 e-/Å2 per single frame). A total of 5521 movie stacks was collected. Raw cryo-EM data preprocessing was performed with Warp software (Tegunov et al., 2019). All further data processing steps were performed using the cryoSPARC v3.2.0 software (Punjani et al., 2017). Results: For final cryo-EM map refinement, 140,000 particles were used resulting in 2.7 Å resolution estimated using an FSC=0.143 gold-standard threshold. The obtained structural data clearly demonstrate the peculiarities of the spatial organization of the 40S ribosomal subunit, like the motility of the head relative to the body revealed by 3D variability analysis. Conclusion: The resulting structure was solved at a significantly higher resolution compared to the previously published structure of a plant ribosome (Armache et al., 2010) and will be used as a reference for further studies of translation initiation in plants.

A self-supervised workflow for particle picking in cryo-EM.

High-resolution single-particle cryo-EM data analysis relies on accurate particle picking. To facilitate the particle picking process, a self-supervised workflow has been developed. This includes an iterative strategy, which uses a 2D class average to improve training particles, and a progressively improved convolutional neural network for particle picking. To automate the selection of particles, a threshold is defined (%/Res) using the ratio of percentage class distribution and resolution as a cutoff. This workflow has been tested using six publicly available data sets with different particle sizes and shapes, and can automatically pick particles with minimal user input. The picked particles support high-resolution reconstructions at 3.0 Å or better. This workflow is a step towards automated single-particle cryo-EM data analysis at the stage of particle picking. It may be used in conjunction with commonly used single-particle analysis packages such as Relion, cryoSPARC, cisTEM, SPHIRE and EMAN2.

Unsupervised particle sorting for high-resolution single-particle cryo-EM

Single-particle cryo-electron microscopy (EM) has become a popular technique for determining the structure of challenging biomolecules that are inaccessible to other technologies. Recent advances in automation, both in data collection and data processing, have significantly lowered the barrier for non-expert users to successfully execute the structure determination workflow. Many critical data processing steps, however, still require expert user intervention in order to converge to the correct high-resolution structure. In particular, strategies to identify homogeneous populations of particles rely heavily on subjective criteria that are not always consistent or reproducible among different users. Here, we explore the use of unsupervised strategies for particle sorting that are compatible with the autonomous operation of the image processing pipeline. More specifically, we show that particles can be successfully sorted based on a simple statistical model for the distribution of scores assigned during refinement. This represents an important step towards the development of automated workflows for protein structure determination using single-particle cryo-EM.

Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV

Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination of well-behaved specimens to high-resolution, there nonetheless remains a discrepancy between the resolutions attained for biological specimens and the information limits of modern transmission electron microscopes (TEMs). Instruments operating at 300 kV equipped with DEDs are the current paradigm for high-resolution single-particle cryo-EM, while 200 kV TEMs remain comparatively underutilized for purposes beyond sample screening. Here, we expand upon our prior work and demonstrate that one such 200 kV microscope, the Talos Arctica, equipped with a K2 DED is capable of determining structures of macromolecules to as high as ∼1.7 Å resolution. At this resolution, ordered water molecules are readily assigned and holes in aromatic residues can be clearly distinguished in the reconstructions. This work emphasizes the utility of 200 kV electrons for high-resolution single-particle cryo-EM and applications such as structure-based drug design.

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.