cryo-EM Images Research Articles

Constrained Cyclic Coordinate Descent for Cryo-EM Images at Medium Resolutions: Beyond the Protein Loop Closure Problem.

The cyclic coordinate descent (CCD) method is a popular loop closure method in protein structure modeling. It is a robotics algorithm originally developed for inverse kinematic applications. We demonstrate an effective method of building the backbone of protein structure models using the principle of CCD and a guiding trace. For medium-resolution 3-dimensional (3D) images derived using cryo-electron microscopy (cryo-EM), it is possible to obtain guiding traces of secondary structures and their skeleton connections. Our new method, constrained cyclic coordinate descent (CCCD), builds α-helices, β-strands, and loops quickly and fairly accurately along predefined traces. We show that it is possible to build the entire backbone of a protein fairly accurately when the guiding traces are accurate. In a test of 10 proteins, the models constructed using CCCD show an average of 3.91Å of backbone root mean square deviation (RMSD). When the CCCD method is incorporated in a simulated annealing framework to sample possible shift, translation, and rotation freedom, the models built with the true topology were ranked high on the list, with an average backbone RMSD100 of 3.76Å. CCCD is an effective method for modeling atomic structures after secondary structure traces and skeletons are extracted from 3D cryo-EM images.

Denoising and covariance estimation of single particle cryo-EM images.

The problem of image restoration in cryo-EM entails correcting for the effects of the Contrast Transfer Function (CTF) and noise. Popular methods for image restoration include 'phase flipping', which corrects only for the Fourier phases but not amplitudes, and Wiener filtering, which requires the spectral signal to noise ratio. We propose a new image restoration method which we call 'Covariance Wiener Filtering' (CWF). In CWF, the covariance matrix of the projection images is used within the classical Wiener filtering framework for solving the image restoration deconvolution problem. Our estimation procedure for the covariance matrix is new and successfully corrects for the CTF. We demonstrate the efficacy of CWF by applying it to restore both simulated and experimental cryo-EM images. Results with experimental datasets demonstrate that CWF provides a good way to evaluate the particle images and to see what the dataset contains even without 2D classification and averaging.

Cryo-EM Visualization of Nanobubbles in Aqueous Solutions.

The detection of nanobubbles on surfaces is well established (e.g., AFM and optical microscopy methods), but currently no methods exist for the direct detection of bulk nanobubbles. Here, cryo-electron microscopy (cryo-EM) has been employed to observe bubbles in aqueous solutions for the first time. Nitrogen bubbles generated by a chemical reaction were observed in amorphous ice trapped between two carbon films. The cryo-EM images of bubbles showed the same features as predicted by theory. The fact that no bubbles were observed near an air-water interface suggests that bubbles may diffuse to the nearby air-water interface and escape. The estimate of the bubble diffusion coefficient is about 30-250 μm2/s.

An Effective Computational Method Incorporating Multiple Secondary Structure Predictions in Topology Determination for Cryo-EM Images.

A key idea in de novo modeling of a medium-resolution density image obtained from cryo-electron microscopy is to compute the optimal mapping between the secondary structure traces observed in the density image and those predicted on the protein sequence. When secondary structures are not determined precisely, either from the image or from the amino acid sequence of the protein, the computational problem becomes more complex. We present an efficient method that addresses the secondary structure placement problem in presence of multiple secondary structure predictions and computes the optimal mapping. We tested the method using 12 simulated images from α-proteins and two Cryo-EM images of α-β proteins. We observed that the rank of the true topologies is consistently improved by using multiple secondary structure predictions instead of a single prediction. The results show that the algorithm is robust and works well even when errors/misses in the predicted secondary structures are present in the image or the sequence. The results also show that the algorithm is efficient and is able to handle proteins with as many as 33 helices.

Real-time and decision taking selection of single-particles during automated cryo-EM sessions based on neuro-fuzzy method

Cryo-electron microscopy (cryo-EM) is a three-dimensional (3D) averaging technique that makes use of two-dimensional (2D) images of biological macromolecules preserved in a thin layer of vitreous ice. Recent advances in the field have facilitated the evolution of cryo-EM towards atomic resolution, and the technique provides 3D maps with detailed description of biological macromolecules. Data acquisition at the transmission electron microscope (TEM) is the first crucial step during the single-particle analysis workflow in cryo-EM. In order to exploit the potential of this structural technique for atomic or near-atomic resolution, the initial collection must allow recording of large datasets and, hence, requires operating the TEM in automated mode. The quality of the acquired dataset relies, however, on the expertise of researchers and unsupervised operations might result in low data quality. This work presents the first expert system integrated in a novel scheme to automate cryo-EM data acquisition in a TEM. This development takes advantage of fuzzy logic systems to integrate the working mode of an expert in a linguistic manner and to learn from acquired data through an adaptive network. A new method based on different image-processing algorithms and on adaptive neuro-fuzzy inference systems (ANFIS) identifies, in an unsupervised manner, the single-particles present in cryo-EM images during the automated acquisition on a TEM. This single-particle identification system is integrated in a new intelligent control scheme to automate cryo-EM data acquisition. A classic fuzzy inference system (FIS) was programmed to make appropriate decisions during the session. The designed system can be trained for a specific sample and allows for unsupervised but efficient data collection imitating the working mode of an experienced microscopist.

In Silico Reduction of Conformational Variance in cryo-EM Imaging

One of the biggest challenges in the analysis of cryo-EM images is the heterogeneity and flexibility of the molecules, which severely limits the achievable resolution. We will present our recent work on the development of a computational approach to reduce the conformational variance of a set of single-particle images with the goal of increasing the resolution. We first reconstruct the conformational variance of a molecule from the variance of the density. The information on conformational variance is then used to extensively classify the images into a number of different classes. In a next step, the individual 3D density reconstructions from all classes are recombined into one single (higher resolution) reconstruction by a novel flexible averaging procedure. In the flexible averaging the density grids of two maps are elastically deformed to account for large scale conformational differences thereby reducing the conformational variance of a data set. The goal is to improve the resolution and at the same time to gain a complete picture of the conformational variance of a macromolecule.

Uncovering the Mechanism of Mitofusin 1 through Structural Studies

A balance between mitochondrial membrane fusion and fission is required for normal mitochondrial morphology and function. Mitofusins are outer mitochondrial trans-membrane proteins that are required for mitochondrial fusion and are members of the dynamin family of GTPases. Our goal is to reveal the mechanism of mitofusins by cryo-electron microscopy and biophysical assays. To accomplish this we are studying the structure of mitofusin 1 in a lipid bilayer as well as the function of a soluble truncation. Here, we present a purification strategy of mitofusin 1 and cryo-EM images of full-length mitofusin 1 in a lipid bilayer. In addition, we show that mitofusin 1 alone in the presence of GTP is capable of tethering proteolipsosomes and that the soluble G-domain truncation is enzymatically active. These preliminary findings imply that mitofusins are sufficient for initiating membrane fusion.

Higher Resolution with the Same Data - Tuning cryo-EM Refinement in Relion

Structure refinement used to be limited by crystallization or computational time. We have eliminated the computational burden of cryo-em by accelerating data processing through development of a new, GPU-enabled version of RELION, in collaboration with its ongoing development. The iterative refinement of bio-molecular structures from cryo-em image data, is therefore faster than ever before. However no systematic analysis has been published on the optimal use of input data in the final stages of high-resolution reconstruction. As a consequence, selective exclusion of available image data (at the discretion of the experimenter) represents the currently most efficient (and often used) way to increase the attained resolution. We show that a higher level of reconstructed detail can be achieved by incorporating new methods into existing cryo-em refinement algorithms, which aim to increase the density of high-resolution data used in the final reconstruction steps. Using a combination of tunable parameters and guided data exclusion, we show that these methods can both increase resolution, as well as reduce sensitivity on the input data. These measures facilitate an improvement to the cryo-em refinement method by a) making reconstructed detail more robust to the used data, and b) rendering the process less subjective. Since cryo-em is rapidly becoming a common tool in structural biology, this represents a necessary development for an enormously influential method over the coming years.

A Technical Note on Hilbert Spaces as Mathematical Tools to Probe and Process Cryo-EM Images – An Architectural and Computational Point of View Using Higher Order Logic (HOL)/Scala/Java/JVM Software Environment

‘Hilbert space is where wave functions dwell,’ according to David Hilbert. Therefore, it is both an interesting and inspiring statement to consider and use Hilbert spaces as mathematical tools to process Cryo-electron microscopy (Cryo-EM) images using novel informatics and computing methodologies. In general, Hilbert spaces and their implementation mechanisms could be used in a wider range of science and technological domains. Here, it is proposed to consider HS (Hilbert Spaces), from an imaging processing viewpoint using higher order logic (HOL)/Scala/Java/JVM Software Environment. In this context, a general informatics framework and its computing aspects are discussed. Though Cryo-EM image processing is focused in this technical note, the concepts apply to other domains like transmission electron microscopy/scanning electron microscopy and others. Hilbert spaces are an excellent form of promising mathematical anticipatory tool to process signals. HOL is a widely used tool for creating formal specifications of systems. HOL has been used in both the industry and academia to support formal reasoning in various domains of science and technology, in addition to hardware and software verification. In principle, they could be used to support any project which needs to be defined on a sound mathematical basis. Given the scope of Cryo-EM Imaging Software Systems, it is very much logical to start a computational framework using HOL as the prototyping platform.

Lattice filter for processing image data of three-dimensional protein nanocrystals.

When 300 kV cryo-EM images at Scherzer focus are acquired from ∼ 100 nm thick three-dimensional protein nanocrystals using a Falcon 2 direct electron detector, Fourier transformation can reveal the crystalline lattice to surprisingly high resolutions, even though the images themselves seem to be devoid of any contrast. Here, it is reported how this lattice information can be enhanced by means of a wave finder in combination with Wiener-type maximum-likelihood filtering. This procedure paves the way towards full three-dimensional structure determination at high resolution for protein crystals.

Effect of the viral protease on the dynamics of bacteriophage HK97 maturation intermediates characterized by variance analysis of cryo EM particle ensembles.

Cryo EM structures of maturation-intermediate Prohead I of bacteriophage HK97 with (PhI(Pro+)) and without (PhI(Pro-)) the viral protease packaged have been reported (Veesler et al., 2014). In spite of PhI(Pro+) containing an additional ∼ 100 × 24 kD of protein, the two structures appeared identical although the two particles have substantially different biochemical properties, e.g., PhI(Pro-) is less stable to disassembly conditions such as urea. Here the same cryo EM images are used to characterize the spatial heterogeneity of the particles at 17Å resolution by variance analysis and show that PhI(Pro-) has roughly twice the standard deviation of PhI(Pro+). Furthermore, the greatest differences in standard deviation are present in the region where the δ-domain, not seen in X-ray crystallographic structures or fully seen in cryo EM, is expected to be located. Thus presence of the protease appears to stabilize the δ-domain which the protease will eventually digest.

Robust w-Estimators for Cryo-EM Class Means.

A critical step in cryogenic electron microscopy (cryo-EM) image analysis is to calculate the average of all images aligned to a projection direction. This average, called the class mean, improves the signal-to-noise ratio in single-particle reconstruction. The averaging step is often compromised because of the outlier images of ice, contaminants, and particle fragments. Outlier detection and rejection in the majority of current cryo-EM methods are done using cross-correlation with a manually determined threshold. Empirical assessment shows that the performance of these methods is very sensitive to the threshold. This paper proposes an alternative: a w-estimator of the average image, which is robust to outliers and which does not use a threshold. Various properties of the estimator, such as consistency and influence function are investigated. An extension of the estimator to images with different contrast transfer functions is also provided. Experiments with simulated and real cryo-EM images show that the proposed estimator performs quite well in the presence of outliers.

Principles of cryo-EM single-particle image processing.

Single-particle reconstruction is the process by which 3D density maps are obtained from a set of low-dose cryo-EM images of individual macromolecules. This review considers the fundamental principles of this process and the steps in the overall workflow for single-particle image processing. Also considered are the limits that image signal-to-noise ratio places on resolution and the distinguishing of heterogeneous particle populations.

Ultrastable gold substrates: Properties of a support for high-resolution electron cryomicroscopy of biological specimens.

Electron cryomicroscopy (cryo-EM) allows structure determination of a wide range of biological molecules and specimens. All-gold supports improve cryo-EM images by reducing radiation-induced motion and image blurring. Here we compare the mechanical and electrical properties of all-gold supports to amorphous carbon foils. Gold supports are more conductive, and have suspended foils that are not compressed by differential contraction when cooled to liquid nitrogen temperatures. These measurements show how the choice of support material and geometry can reduce specimen movement by more than an order of magnitude during low-dose imaging. We provide methods for fabrication of all-gold supports and preparation of vitrified specimens. We also analyse illumination geometry for optimal collection of high resolution, low-dose data. Together, the support structures and methods herein can improve the resolution and quality of images from any electron cryomicroscope.

Comparison of an Atomic Model and Its Cryo-EM Image at the Central Axis of a Helix.

Cryo-electron microscopy (cryo-EM) is an important biophysical technique that produces three-dimensional (3D) density maps at different resolutions. Because more and more models are being produced from cryo-EM density maps, validation of the models is becoming important. We propose a method for measuring local agreement between a model and the density map using the central axis of the helix. This method was tested using 19 helices from cryo-EM density maps between 5.5 Å and 7.2 Å resolution and 94 helices from simulated density maps. This method distinguished most of the well-fitting helices, although challenges exist for shorter helices.

A new protocol to accurately determine microtubule lattice seam location.

Microtubules (MTs) are cylindrical polymers of αβ-tubulin that display pseudo-helical symmetry due to the presence of a lattice seam of heterologous lateral contacts. The structural similarity between α- and β-tubulin makes it difficult to computationally distinguish them in the noisy cryo-EM images, unless a marker protein for the tubulin dimer, such as kinesin motor domain, is present. We have developed a new data processing protocol that can accurately determine αβ-tubulin register and seam location for MT segments. Our strategy can deal with difficult situations, where the marker protein is relatively small or the decoration of marker protein is sparse. Using this new seam-search protocol, combined with movie processing for data from a direct electron detection camera, we were able to determine the cryo-EM structures of MT at 3.5 Å resolution in different functional states. The successful distinction of α- and β-tubulin allowed us to visualize the nucleotide state at the E-site and the configuration of lateral contacts at the seam.

The U4 Antibody Epitope on Human Papillomavirus 16 Identified by Cryo-electron Microscopy.

The human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T=7 icosahedral capsid. H16.U4 is a type-specific monoclonal antibody recognizing a conformation-dependent neutralizing epitope of HPV thought to include the L1 protein C terminus. The structure of human papillomavirus 16 (HPV16) complexed with H16.U4 fragments of antibody (Fab) was solved by cryo-electron microscopy (cryo-EM) image reconstruction. Atomic structures of virus and Fab were fitted into the corresponding cryo-EM densities to identify the antigenic epitope. The antibody footprint mapped predominately to the L1 C-terminal arm with an additional contact point on the side of the capsomer. This footprint describes an epitope that is presented capsid-wide. However, although the H16.U4 epitope suggests the presence of 360 potential binding sites exposed in the capsid valley between each capsomer, H16.U4 Fab bound only to epitopes located around the icosahedral five-fold vertex of the capsid. Thus, the binding characteristics of H16.U4 defined in this study showed a distinctive selectivity for local conformation-dependent interactions with specific L1 invading arms between five-fold related capsomers. Human papillomavirus 16 (HPV16) is the most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the structures of the HPV16 capsid complexes using H16.U4 fragment of antibody (Fab). Different from most other antibodies directed against surface loops, H16.U4 monoclonal antibody is unique in targeting the C-terminal arm of the L1 protein. This monoclonal antibody (MAb) is used throughout the HPV research community in HPV serological and vaccine development and to define mechanisms of HPV uptake. The unique binding mode of H16.U4 defined here shows important conformation-dependent interactions within the HPV16 capsid. By targeting an important structural and conformational epitope, H16.U4 may identify subtle conformational changes in different maturation stages of the HPV capsid and provide a key probe to analyze the mechanisms of HPV uptake during the early stages of virus infection. Our analyses precisely define important conformational epitopes on HPV16 capsids that are key targets for successful HPV prophylactic vaccines.

Asynchronous data acquisition and on-the-fly analysis of dose fractionated cryoEM images by UCSFImage.

Newly developed direct electron detection cameras have a high image output frame rate that enables recording dose fractionated image stacks of frozen hydrated biological samples by electron cryomicroscopy (cryoEM). Such novel image acquisition schemes provide opportunities to analyze cryoEM data in ways that were previously impossible. The file size of a dose fractionated image stack is 20-60 times larger than that of a single image. Thus, efficient data acquisition and on-the-fly analysis of a large number of dose-fractionated image stacks become a serious challenge to any cryoEM data acquisition system. We have developed a computer-assisted system, named UCSFImage4, for semi-automated cryo-EM image acquisition that implements an asynchronous data acquisition scheme. This facilitates efficient acquisition, on-the-fly motion correction, and CTF analysis of dose fractionated image stacks with a total time of ∼60s/exposure. Here we report the technical details and configuration of this system.

Self-Assembly of an Alphavirus Core-like Particle Is Distinguished by Strong Intersubunit Association Energy and Structural Defects.

Weak association energy can lead to uniform nanostructures: defects can anneal due to subunit lability. What happens when strong association energy leads to particles where defects are trapped? Alphaviruses are enveloped viruses whose icosahedral nucleocapsid core can assemble independently. We used a simplest case system to study Ross River virus (RRV) core-like particle (CLP) self-assembly using purified capsid protein and a short DNA oligomer. We find that capsid protein binds the oligomer with high affinity to form an assembly competent unit (U). Subsequently, U assembles with concentration dependence into CLPs. We determined that U-U pairwise interactions are very strong (ca. -6 kcal/mol) compared to other virus assembly systems. Assembled RRV CLPs appeared morphologically uniform and cryo-EM image reconstruction with imposed icosahedral symmetry yielded a T = 4 structure. However, 2D class averages of the CLPs show that virtually every class had disordered regions. These results suggested that irregular cores may be present in RRV virions. To test this hypothesis, we determined 2D class averages of RRV virions using authentic virions or only the core from intact virions isolated by computational masking. Virion-based class averages were symmetrical, geometric, and corresponded well to projections of image reconstructions. In core-based class averages, cores and envelope proteins in many classes were disordered. These results suggest that partly disordered components are common even in ostensibly well-ordered viruses, a biological realization of a patchy particle. Biological advantages of partly disordered complexes may arise from their ease of dissociation and asymmetry.

Description and comparison of algorithms for correcting anisotropic magnification in cryo-EM images.

Single particle electron cryomicroscopy (cryo-EM) allows for structures of proteins and protein complexes to be determined from images of non-crystalline specimens. Cryo-EM data analysis requires electron microscope images of randomly oriented ice-embedded protein particles to be rotated and translated to allow for coherent averaging when calculating three-dimensional (3D) structures. Rotation of 2D images is usually done with the assumption that the magnification of the electron microscope is the same in all directions. However, due to electron optical aberrations, this condition is not met with some electron microscopes when used with the settings necessary for cryo-EM with a direct detector device (DDD) camera. Correction of images by linear interpolation in real space has allowed high-resolution structures to be calculated from cryo-EM images for symmetric particles. Here we describe and compare a simple real space method, a simple Fourier space method, and a somewhat more sophisticated Fourier space method to correct images for a measured anisotropy in magnification. Further, anisotropic magnification causes contrast transfer function (CTF) parameters estimated from image power spectra to have an apparent systematic astigmatism. To address this problem we develop an approach to adjust CTF parameters measured from distorted images so that they can be used with corrected images. The effect of anisotropic magnification on CTF parameters provides a simple way of detecting magnification anisotropy in cryo-EM datasets.