Single-particle cryo-EM Reconstruction Research Articles

CryoEM Single Particle Reconstruction of Dephosphorylated HMM from Smooth Muscle

The asymmetric head-head interaction that characterizes the inhibited state of smooth muscle myosin was first discovered in 3-D images of 2-D arrays formed on lipid monolayers at ∼2 nm resolution. The conformation is generally referred to as the “interacting heads motif” or IHM and has since been observed in 3-D images of thick filaments from all species examined from vertebrate striated muscle to the flight muscles of the large waterbug Lethocerus sp. indicating it is a ubiquitous myosin II inhibited conformation. However, current 3-D images reported from filaments are limited to resolutions lower than 1 nm. Here we report on progress toward an atomic resolution structure of the IHM of smooth muscle HMM using single particle methods from dispersions of single particles bound to a lipid monolayer. Data were collected on a Titan Krios equipped with a Volta Phase Plate and a Direct Electron DE-64 detector. Initial reconstructions, starting from a total of 1464 phase plate images from which 588,000 particles were selected using Gautomatch, which is a template matching particle picker, using a template derived from the smooth muscle HMM crystal structure. This structure went to 1.49 nm resolution from a subset of 285300 particles. The free and blocked heads of the IHM are easily distinguishable but the S2 domain is not yet visible. The structure is being refined with addition of new images. Supported by NIH.

PIXER: an automated particle-selection method based on segmentation using a deep neural network

BackgroundCryo-electron microscopy (cryo-EM) has become a widely used tool for determining the structures of proteins and macromolecular complexes. To acquire the input for single-particle cryo-EM reconstruction, researchers must select hundreds of thousands of particles from micrographs. As the signal-to-noise ratio (SNR) of micrographs is extremely low, the performance of automated particle-selection methods is still unable to meet research requirements. To free researchers from this laborious work and to acquire a large number of high-quality particles, we propose an automated particle-selection method (PIXER) based on the idea of segmentation using a deep neural network.ResultsFirst, to accommodate low-SNR conditions, we convert micrographs into probability density maps using a segmentation network. These probability density maps indicate the likelihood that each pixel of a micrograph is part of a particle instead of just background noise. Particles selected from density maps have a more robust signal than do those directly selected from the original noisy micrographs. Second, at present, there is no segmentation-training dataset for cryo-EM. To enable our plan, we present an automated method to generate a training dataset for segmentation using real-world data. Third, we propose a grid-based, local-maximum method to locate the particles from the probability density maps. We tested our method on simulated and real-world experimental datasets and compared PIXER with the mainstream methods RELION, DeepEM and DeepPicker to demonstrate its performance. The results indicate that, as a fully automated method, PIXER can acquire results as good as the semi-automated methods RELION and DeepEM.ConclusionTo our knowledge, our work is the first to address the particle-selection problem using the segmentation network concept. As a fully automated particle-selection method, PIXER can free researchers from laborious particle-selection work. Based on the results of experiments, PIXER can acquire accurate results under low-SNR conditions within minutes.

Structures of Mycobacterium smegmatis 70S ribosomes in complex with HPF, tmRNA, and P-tRNA

Ribosomes are the dynamic protein synthesis machineries of the cell. They may exist in different functional states in the cell. Therefore, it is essential to have structural information on these different functional states of ribosomes to understand their mechanism of action. Here, we present single particle cryo-EM reconstructions of the Mycobacterium smegmatis 70S ribosomes in the hibernating state (with HPF), trans-translating state (with tmRNA), and the P/P state (with P-tRNA) resolved to 4.1, 12.5, and 3.4 Å, respectively. A comparison of the P/P state with the hibernating state provides possible functional insights about the Mycobacteria-specific helix H54a rRNA segment. Interestingly, densities for all the four OB domains of bS1 protein is visible in the hibernating 70S ribosome displaying the molecular details of bS1-70S interactions. Our structural data shows a Mycobacteria-specific H54a-bS1 interaction which seems to prevent subunit dissociation and degradation during hibernation without the formation of 100S dimer. This indicates a new role of bS1 protein in 70S protection during hibernation in Mycobacteria in addition to its conserved function during translation initiation.

High resolution single particle cryo-electron microscopy using beam-image shift.

Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images. For optimal efficiency, automated data acquisition software packages typically employ some beam-image shift targeting as this method is both fast and accurate (±0.1 µm). In contrast, using only stage movement, relocation to a targeted area under low-dose conditions can only be achieved in combination with multiple iterations or long relaxation times, both reducing efficiency. Nevertheless it is well known that applying beam-image shift induces beam-tilt and with it a potential structure phase error with a phase error π/4 the highest acceptable value. This theory has been used as an argument against beam-image shift for high resolution data collection. Nevertheless, in practice many small beam-image shift datasets have resulted in 3D reconstructions beyond the π/4 phase error limit. To address this apparent contradiction, we performed cryo-EM single-particle reconstructions on a T20S proteasome sample using applied beam-image shifts corresponding to beam tilts from 0 to 10 mrad. To evaluate the results we compared the FSC values, and examined the water density peaks in the 3D map. We conclude that the phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit.

Map Challenge assessment: Fair comparison of single particle cryoEM reconstructions.

Cryo-electron microscopy (cryoEM) is capable of achieving near-atomic resolution of biomolecular structures due to recent advances in hardware. Despite the long history of image processing software development for cryoEM, uncertainty about best practices and validation remains. The Map Challenge was therefore designed to test the current state of single particle reconstruction. As the first such challenge, the participants were given the freedom to analyze the cases in whichever way they wanted. Therefore, the maps submitted feature different sizes, sampling and orientations, making assessment non-trivial. To be fair, I developed a method to pose all maps in each case in the same configuration with minimal interpolation. I assessed the quality of these maps by visual inspection and Fourier shell correlation (FSC). Comparing the even-odd FSC with an FSC calculated against a reference structure analysis, I concluded that the quality of the maps related more to the user than to other factors, such as the software package used. Poor quality maps suffer either from lack of data or poor choices made by the user. Some maps appear significantly better than a reference or consensus of other maps, indicating overfitting. Best practices to avoid problems include an understanding of the effects of reference map modifications on particle image alignment, and generating appropriate masks. Ultimately, none of the issues revealed in the Map Challenge is insurmountable, as underscored by the excellent quality of reconstructions achieved by a significant number of participants.

Cryo-EM structure of human mitochondrial trifunctional protein

The mitochondrial trifunctional protein (TFP) catalyzes three reactions in the fatty acid β-oxidation process. Mutations in the two TFP subunits cause mitochondrial trifunctional protein deficiency and acute fatty liver of pregnancy that can lead to death. Here we report a 4.2-Å cryo-electron microscopy α2β2 tetrameric structure of the human TFP. The tetramer has a V-shaped architecture that displays a distinct assembly compared with the bacterial TFPs. A concave surface of the TFP tetramer interacts with the detergent molecules in the structure, suggesting that this region is involved in associating with the membrane. Deletion of a helical hairpin in TFPβ decreases its binding to the liposomes in vitro and reduces its membrane targeting in cells. Our results provide the structural basis for TFP function and have important implications for fatty acid oxidation related diseases.

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope

Following the recent emergence of Zika virus (ZIKV), many murine and human neutralizing anti-ZIKV antibodies have been reported. Given the risk of virus escape mutants, engineering antibodies that target mutationally constrained epitopes with therapeutically relevant potencies can be valuable for combating future outbreaks. Here, we applied computational methods toengineer an antibody, ZAb_FLEP, that targets a highly networked and therefore mutationally constrained surface formed by the envelope protein dimer. ZAb_FLEP neutralized a breadth of ZIKV strains and protected mice in distinct invivo models, including resolving vertical transmission and fetal mortality in infected pregnant mice. Serial passaging of ZIKV in the presence of ZAb_FLEP failed to generate viral escape mutants, suggesting that its epitope is indeed mutationally constrained. A single-particle cryo-EM reconstruction of the Fab-ZIKV complex validated the structural model and revealed insights into ZAb_FLEP's neutralization mechanism. ZAb_FLEP has potential as a therapeutic in future outbreaks.

Pushing the resolution limit by correcting the Ewald sphere effect in single-particle Cryo-EM reconstructions

The Ewald sphere effect is generally neglected when using the Central Projection Theorem for cryo electron microscopy single-particle reconstructions. This can reduce the resolution of a reconstruction. Here we estimate the attainable resolution and report a “block-based” reconstruction method for extending the resolution limit. We find the Ewald sphere effect limits the resolution of large objects, especially large viruses. After processing two real datasets of large viruses, we show that our procedure can extend the resolution for both datasets and can accommodate the flexibility associated with large protein complexes.

Alternate binding modes of anti-CRISPR viral suppressors AcrF1/2 to Csy surveillance complex revealed by cryo-EM structures.

Bacteriophages encode anti-CRISPR suppressors to counteract the CRISPR/Cas immunity of their bacterial hosts, thus facilitating their survival and replication. Previous studies have shown that two phage-encoded anti-CRISPR proteins, AcrF1 and AcrF2, suppress the type I-F CRISPR/Cas system of Pseudomonas aeruginosa by preventing target DNA recognition by the Csy surveillance complex, but the precise underlying mechanism was unknown. Here we present the structure of AcrF1/2 bound to the Csy complex determined by cryo-EM single-particle reconstruction. By structural analysis, we found that AcrF1 inhibits target DNA recognition of the Csy complex by interfering with base pairing between the DNA target strand and crRNA spacer. In addition, multiple copies of AcrF1 bind to the Csy complex with different modes when working individually or cooperating with AcrF2, which might exclude target DNA binding through different mechanisms. Together with previous reports, we provide a comprehensive working scenario for the two anti-CRISPR suppressors, AcrF1 and AcrF2, which silence CRISPR/Cas immunity by targeting the Csy surveillance complex.

Precise and Reversible Protein-Microtubule-Like Structure with Helicity Driven by Dual Supramolecular Interactions.

Protein microtubule is a significant self-assembled architecture found in nature with crucial biological functions. However, mimicking protein microtubules with precise structure and controllable self-assembly behavior remains highly challenging. In this work, we demonstrate that by using dual supramolecular interactions from a series of well-designed ligands, i.e., protein-sugar interaction and π-π stacking, highly homogeneous protein microtubes were achieved from tetrameric soybean agglutinin without any chemical or biological modification. Using combined cryo-EM single-particle reconstruction and computational modeling, the accurate structure of protein microtube was determined. The helical protein microtube is consisted of three protofilaments, each of which features an array of soybean agglutinin tetramer linked by the designed ligands. Notably, the microtubes resemble the natural microtubules in their structural and dynamic features such as the shape and diameter and the controllable and reversible assembly behavior, among others. Furthermore, the protein microtubes showed an ability to enhance immune response, demonstrating its great potential for biological applications.

C3-O-04Functional structures of ion channels in lipid environment

The structural study of membrane protein is getting more and more popular as for the importance in its function especially for drug discovery. Recent progress in structure and function relationship of membrane proteins is based on the near atomic structures obtained by X-ray crystallography. Recently, cryo-EM single particle reconstruction of membrane proteins has been reported in such a resolution range due to the benefit of direct electron detector and high performance computation [ 1 , 2 ]. Even though those are useful to understand the structure and function relationship, it would be important to remind that there are in detergent solubilized state. The final goal of structural study of membrane protein is to utilize the membrane proteins in lipid environment. We have developed alternative methods so-called Random Spherically-Constrained (RSC) reconstruction in which membrane proteins on liposome is directly imaged in cryo-EM [ 3 ]. We have struggled a lot to optimize sample preparation, specimen preparation, imaging condition and image processing algorithm and then, now we have achieved sub-nanometer resolution reconstruction of potassium channel, Kv1.2-beta2.1 in detergent free, lipid environment.

Single-particle cryoEM analysis at near-atomic resolution from several thousand asymmetric subunits.

A single-particle cryoEM reconstruction of the large ribosomal subunit from Saccharomyces cerevisiae was obtained from a dataset of ∼75,000 particles. The gold-standard and frequency-limited approaches to single-particle refinement were each independently used to determine orientation parameters for the final reconstruction. Both approaches showed similar resolution curves and nominal resolution values for the 60S dataset, estimated at 2.9 Å. The amount of over-fitting present during frequency-limited refinement was quantitatively analyzed using the high-resolution phase-randomization test, and the results showed no apparent over-fitting. The number of asymmetric subunits required to reach specific resolutions was subsequently analyzed by refining subsets of the data in an ab initio manner. With our data collection and processing strategies, sub-nanometer resolution was obtained with ∼200 asymmetric subunits (or, equivalently for the ribosomal subunit, particles). Resolutions of 5.6 Å, 4.5 Å, and 3.8 Å were reached with ∼1000, ∼1600, and ∼5000 asymmetric subunits, respectively. At these resolutions, one would expect to detect alpha-helical pitch, separation of beta-strands, and separation of Cα atoms, respectively. Using this map, together with strategies for ab initio model building and model refinement, we built a region of the ribosomal protein eL6, which was missing in previous models of the yeast ribosome. The relevance for more routine high-resolution structure determination is discussed.

Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6.

Biological specimens suffer radiation damage when imaged in an electron microscope, ultimately limiting the attainable resolution. At a given resolution, an optimal exposure can be defined that maximizes the signal-to-noise ratio in the image. Using a 2.6 Å resolution single particle cryo-EM reconstruction of rotavirus VP6, determined from movies recorded with a total exposure of 100 electrons/Å(2), we obtained accurate measurements of optimal exposure values over a wide range of resolutions. At low and intermediate resolutions, our measured values are considerably higher than obtained previously for crystalline specimens, indicating that both images and movies should be collected with higher exposures than are generally used. We demonstrate a method of using our optimal exposure values to filter movie frames, yielding images with improved contrast that lead to higher resolution reconstructions. This 'high-exposure' technique should benefit cryo-EM work on all types of samples, especially those of relatively low-molecular mass.

SubspaceEM: A fast maximum-a-posteriori algorithm for cryo-EM single particle reconstruction.

Single particle reconstruction methods based on the maximum-likelihood principle and the expectation-maximization (E-M) algorithm are popular because of their ability to produce high resolution structures. However, these algorithms are computationally very expensive, requiring a network of computational servers. To overcome this computational bottleneck, we propose a new mathematical framework for accelerating maximum-likelihood reconstructions. The speedup is by orders of magnitude and the proposed algorithm produces similar quality reconstructions compared to the standard maximum-likelihood formulation. Our approach uses subspace approximations of the cryo-electron microscopy (cryo-EM) data and projection images, greatly reducing the number of image transformations and comparisons that are computed. Experiments using simulated and actual cryo-EM data show that speedup in overall execution time compared to traditional maximum-likelihood reconstruction reaches factors of over 300.

Direct imaging electron microscopy (EM) methods in modern structural biology: overview and comparison with X-ray crystallography and single-particle cryo-EM reconstruction in the studies of large macromolecules.

Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X-ray crystallography and single-particle cryo-electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative-staining, rotary-shadowing and freeze-etching EM, which are categorised here as 'direct imaging EM methods'. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome-translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three-dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo.

Cryo-Electron Microscopy (Cryo-EM) Structure of a Cap-Independent Translational Enhancer of the Turnip Crinkle Virus (TCV) Bound to the Eukaryotic Ribosome

Many plant viruses, unlike animal viruses, use 3′ translational enhancers that function by unknown mechanism to achieve translation initiation. A translational enhancer in the 3′-UTR of turnip crinkle virus (TCV) has been shown to synergistically enhance translation when associated with the TCV 5′-UTR. The major enhancement has been shown, biochemically, due to a T-shaped structure (TSS) which binds to the 80S ribosomes with the aid of a pseudoknot that mimics the tRNA acceptor stem. The research presented here focuses on the structural interaction of the TSS structure with the 80S eukaryotic ribosome from Saccharomyces cerevisiae using the single-particle cryo-EM reconstruction technique. Our preliminary work shows that the density corresponding to the TSS structure is directly visible in the cryo-EM map, and most of the ribosomes containing the TSS structure are in the ratcheted-like intersubunit-rotated state. These results match the biochemical results. We are currently investigating the detailed binding interactions between the TSS and the ribosome. Interpretation of this cryo-EM map is a first step toward an understanding of the molecular mechanism of translation enhancement and regulation by the TSS structure.

Structural diversity of bacterial flagellar motors

The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species.

Low cost, high performance GPU computing solution for atomic resolution cryoEM single-particle reconstruction

Recent advancements in cryo-electron microscopy (cryoEM) have made it technically possible to determine the three-dimensional (3D) structures of macromolecular complexes at atomic resolution. However, processing the large amount of data needed for atomic resolution reconstructions requires either accessing to very expensive computer clusters or waiting for weeks of continuous computation in a personal computer (PC). In this paper, we present a practical computational solution to this 3D reconstruction problem through the optimal utilization of the processing capabilities of both commodity graphics hardware (i.e., general purpose graphics processing unit (GPGPU)). Our solution, which is implemented in a new program, called eLite3D, has a number of advanced features of general interests. First, we construct interleaved schemes to prevent the data race condition intrinsic in merging of 2D data into a 3D volume. Second, we introduce a processing pipeline strategy to optimally balance I/O and computation operations, thus improving CPU and GPGPU parallelism. The speedup of eLite3D is up to 100 times over other commonly used 3D reconstruction programs with the same accuracy, thus allowing completion of atomic resolution 3D reconstructions of large complexes in a PC in 1-2h other than days or weeks. Our result provides a practical solution to atomic resolution cryoEM (asymmetric or symmetric) reconstruction and offers useful guidelines for developing GPGPU applications in general.

Visualizing tmRNA after its accommodation in the Ribosome

In eubacteria, translation of an mRNA that lacks a stop codon produces defective polypeptide that stalls on the ribosome. Transfer-messenger RNA (tmRNA), a molecule in eubacteria that possesses functions of both mRNA and tRNA, rescues the stalled ribosome by “trans-translation,” a process by which the tmRNA is recruited to the ribosome with the help of EF-Tu and small protein B (smpB). Tanslation is resumed on the open reading frame of the mRNA-like domain (MLD) of the tmRNA. Several structures of tmRNA in complex with ribosome, in the accommodating and the accommodated states, have been studied by cryo-EM single-particle reconstruction (Gillet et al., 2007; Kaur et al., 2006; Valle et al., 2003). However, the structures of the complex in the subsequent stages of trans-translation remain unknown. Here, using mutagenesis, we have been able to trap the complex in different stages of trans-translation by substituting one of the sense codons of the MLD open reading frame with a stop codon. Structures of these complexes were obtained by the cryo-EM single particle reconstruction technique. To address the sample heterogeneity, we used the maximum-liklihood classification method (Scheres et al., 2007). The resulting density maps were analyzed by rigid body fitting in combination with biochemical data. We discovered that part of the tmRNA molecule maintains a relative defined structure during tran-translation. Also, we identified several possible binding sites of the tRNA like domain (TLD) of tmRNA and smpB on the ribosome.Gillet et al. (2007) J. Biol. Chem. 282: 6356-6363.Kaur S. et al. (2006) Proc. Natl. Acad. Sci. USA 103:16484-16489.Scheres S. et al. (2007) Nature Methods 4: 27-29.Valle M. et al. (2003) Science 300: 127-130.

A test-bed for optimizing high-resolution single particle reconstructions

It is becoming routine for cryoEM single particle reconstructions to result in 3D electron density maps with resolutions of approximately 10A, but maps with resolutions of 5A or better are still celebrated events. The electron microscope has a resolving power to better than 2A, and thus should not be a limiting factor; instead the practical limitations in resolution most likely arise from a combination of specimen preparation methods, data collection parameters, and data analysis procedures. With the aid of a highly automated system for acquiring images, coupled to a relational database to keep track of all processing parameters, we have taken a systematic approach to optimizing parameters affecting the resolution of single particle reconstructions. Using GroEL as a test-bed, we performed a series of 3D reconstructions where we systematically varied the number of particles used in computing the map, the accelerating voltage of the microscope, and the electron dose used to acquire the images. We also investigated methods for excluding unacceptable or "bad" particles from contributing to the final 3D map. Using relatively standard instrumentation (Tecnai F20, 4K x 4K CCD, side entry cold stage) and a completely automated approach, these approaches resulted in a map with a nominal resolution of 5.4A (FSC(0.5)) in which secondary structure is clearly discernable and the handedness of some of the alpha-helices in the GroEL structure can be determined.