Field Of cryo-EM Research Articles

Towards interpretable Cryo-EM: disentangling latent spaces of molecular conformations.

Molecules are essential building blocks of life and their different conformations (i.e., shapes) crucially determine the functional role that they play in living organisms. Cryogenic Electron Microscopy (cryo-EM) allows for acquisition of large image datasets of individual molecules. Recent advances in computational cryo-EM have made it possible to learn latent variable models of conformation landscapes. However, interpreting these latent spaces remains a challenge as their individual dimensions are often arbitrary. The key message of our work is that this interpretation challenge can be viewed as an Independent Component Analysis (ICA) problem where we seek models that have the property of identifiability. That means, they have an essentially unique solution, representing a conformational latent space that separates the different degrees of freedom a molecule is equipped with in nature. Thus, we aim to advance the computational field of cryo-EM beyond visualizations as we connect it with the theoretical framework of (nonlinear) ICA and discuss the need for identifiable models, improved metrics, and benchmarks. Moving forward, we propose future directions for enhancing the disentanglement of latent spaces in cryo-EM, refining evaluation metrics and exploring techniques that leverage physics-based decoders of biomolecular systems. Moreover, we discuss how future technological developments in time-resolved single particle imaging may enable the application of nonlinear ICA models that can discover the true conformation changes of molecules in nature. The pursuit of interpretable conformational latent spaces will empower researchers to unravel complex biological processes and facilitate targeted interventions. This has significant implications for drug discovery and structural biology more broadly. More generally, latent variable models are deployed widely across many scientific disciplines. Thus, the argument we present in this work has much broader applications in AI for science if we want to move from impressive nonlinear neural network models to mathematically grounded methods that can help us learn something new about nature.

Imaging intracellular components in situ using super-resolution cryo-correlative light and electron microscopy.

Super-resolution cryo-correlative light and electron microscopy (SRcryoCLEM) is emerging as a powerful method to enable targeted in situ structural studies of biological samples. By combining the high specificity and localization accuracy of single-molecule localization microscopy (cryoSMLM) with the high resolution of cryo-electron tomography (cryoET), this method enables accurately targeted data acquisition and the observation and identification of biomolecules within their natural cellular context. Despite its potential, the adaptation of SRcryoCLEM has been hindered by the need for specialized equipment and expertise. In this chapter, we outline a workflow for cryoSMLM and cryoET-based SRcryoCLEM, and we demonstrate that, given the right tools, it is possible to incorporate cryoSMLM into an established cryoET workflow. Using Vimentin as an exemplary target of interest, we demonstrate all stages of an SRcryoCLEM experiment: performing cryoSMLM, targeting cryoET acquisition based on single-molecule localization maps, and correlation of cryoSMLM and cryoET datasets using scNodes, a software package dedicated to SRcryoCLEM. By showing how SRcryoCLEM enables the imaging of specific intracellular components in situ, we hope to facilitate adoption of the technique within the field of cryoEM.

MDSPACE and MDTOMO Software for Extracting Continuous Conformational Landscapes from Datasets of Single Particle Images and Subtomograms Based on Molecular Dynamics Simulations: Latest Developments in ContinuousFlex Software Package.

Cryo electron microscopy (cryo-EM) instrumentation allows obtaining 3D reconstruction of the structure of biomolecular complexes in vitro (purified complexes studied by single particle analysis) and in situ (complexes studied in cells by cryo electron tomography). Standard cryo-EM approaches allow high-resolution reconstruction of only a few conformational states of a molecular complex, as they rely on data classification into a given number of classes to increase the resolution of the reconstruction from the most populated classes while discarding all other classes. Such discrete classification approaches result in a partial picture of the full conformational variability of the complex, due to continuous conformational transitions with many, uncountable intermediate states. In this article, we present the software with a user-friendly graphical interface for running two recently introduced methods, namely, MDSPACE and MDTOMO, to obtain continuous conformational landscapes of biomolecules by analyzing in vitro and in situ cryo-EM data (single particle images and subtomograms) based on molecular dynamics simulations of an available atomic model of one of the conformations. The MDSPACE and MDTOMO software is part of the open-source ContinuousFlex software package (starting from version 3.4.2 of ContinuousFlex), which can be run as a plugin of the Scipion software package (version 3.1 and later), broadly used in the cryo-EM field.

A minority of final stacks yields superior amplitude in single-particle cryo-EM

Cryogenic electron microscopy (cryo-EM) is widely used to determine near-atomic resolution structures of biological macromolecules. Due to the low signal-to-noise ratio, cryo-EM relies on averaging many images. However, a crucial question in the field of cryo-EM remains unanswered: how close can we get to the minimum number of particles required to reach a specific resolution in practice? The absence of an answer to this question has impeded progress in understanding sample behavior and the performance of sample preparation methods. To address this issue, we develop an iterative particle sorting and/or sieving method called CryoSieve. Extensive experiments demonstrate that CryoSieve outperforms other cryo-EM particle sorting algorithms, revealing that most particles are unnecessary in final stacks. The minority of particles remaining in the final stacks yield superior high-resolution amplitude in reconstructed density maps. For some datasets, the size of the finest subset approaches the theoretical limit.

Scipion Flexibility Hub: an integrative framework for advanced analysis of conformational heterogeneity in cryoEM.

Understanding how structure and function meet to drive biological processes is progressively shifting the cryoEM field towards a more advanced analysis of macromolecular flexibility. Thanks to techniques such as single-particle analysis and electron tomography, it is possible to image a macromolecule in different states, information that can subsequently be extracted through advanced image-processing methods to build a richer approximation of a conformational landscape. However, the interoperability of all of these algorithms remains a challenging task that is left to users, preventing them from defining a single flexible workflow in which conformational information can be addressed by different algorithms. Therefore, in this work, a new framework integrated into Scipion is proposed called the Flexibility Hub. This framework automatically handles intercommunication between different heterogeneity software, simplifying the task of combining the software into workflows in which the quality and the amount of information extracted from flexibility analysis is maximized.

ContinuousFlex: Software package for analyzing continuous conformational variability of macromolecules in cryo electron microscopy and tomography data

ContinuousFlex is a user-friendly open-source software package for analyzing continuous conformational variability of macromolecules in cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET) data. In 2019, ContinuousFlex became available as a plugin for Scipion, an image processing software package extensively used in the cryo-EM field. Currently, ContinuousFlex contains software for running (1) recently published methods HEMNMA-3D, TomoFlow, and NMMD; (2) earlier published methods HEMNMA and StructMap; and (3) methods for simulating cryo-EM and cryo-ET data with conformational variability and methods for data preprocessing. It also includes external software for molecular dynamics simulation (GENESIS) and normal mode analysis (ElNemo), used in some of the mentioned methods. The HEMNMA software has been presented in the past, but not the software of other methods. Besides, ContinuousFlex currently also offers a deep learning extension of HEMNMA, named DeepHEMNMA. In this article, we review these methods in the context of the ContinuousFlex package, developed to facilitate their use by the community.

A Transfer Learning-Based Classification Model for Particle Pruning in Cryo-Electron Microscopy.

The cryo-electron microscopy (cryo-EM) single-particle analysis requires tens of thousands of particle projections to reveal structural information of macromolecular complexes. However, due to the low signal-to-noise ratio and the presence of high contrast artifacts and contaminants in the micrographs, the semiautomatic and fully automatic particle picking algorithms tend to suffer from high false-positive rates, which degrades the confidence of structure determination. In this study, we introduce PickerOptimizer (PO), a transfer learning-based classification neural network for particle pruning in cryo-EM, as an additional strategy to complement the current automated particle picking algorithms. To achieve high classification performance with minimal human intervention, we adopted two key strategies: (1) utilizing the transfer learning techniques to train the convolutional neural network, where the knowledge gained from public classification datasets is applied to the field of cryo-EM. (2) Designing a multiloss strategy, a combination of multiple loss functions, to guide the optimization of the network parameters. To reduce the domain shift between cryo-EM images and natural images for pretraining, we build the first image classification dataset for cryo-EM, which contains positive and negative samples collected from EMPIAR entries. The PO is tested on 14 public experimental datasets, achieving accuracy and F1 scores above 95% in most cases. Furthermore, three case studies are provided to verify the model performance by applying PO on problematic particle selections, showing that our algorithm achieved better or comparable performance compared with other particle pruning strategies.

Recent Technical Advances in Sample Preparation for Single-Particle Cryo-EM.

Cryo-sample preparation is a vital step in the process of obtaining high-resolution structures of macromolecules by using the single-particle cryo–electron microscopy (cryo-EM) method; however, cryo-sample preparation is commonly hampered by high uncertainty and low reproducibility. Specifically, the existence of air-water interfaces during the sample vitrification process could cause protein denaturation and aggregation, complex disassembly, adoption of preferred orientations, and other serious problems affecting the protein particles, thereby making it challenging to pursue high-resolution 3D reconstruction. Therefore, sample preparation has emerged as a critical research topic, and several new methods for application at various preparation stages have been proposed to overcome the aforementioned hurdles. Here, we summarize the methods developed for enhancing the quality of cryo-samples at distinct stages of sample preparation, and we offer insights for developing future strategies based on diverse viewpoints. We anticipate that cryo-sample preparation will no longer be a limiting step in the single-particle cryo-EM field as increasing numbers of methods are developed in the near future, which will ultimately benefit the entire research community.

Waffle Method: A general and flexible approach for improving throughput in FIB-milling

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens with vitrification issues, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and specimens that distribute poorly across grid squares. Here we present a general approach called the ‘Waffle Method’ which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. We propose the Waffle Method as a way to achieve many advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.

Alternative preservation methods in cryo-EM to overcome challenging specimens

In the past decade, many developments have advanced the cryo-EM field in imaging technologies and processing methodologies. Still, there are many challenging samples that cannot be structurally characterized with conventional cryo-EM preparation procedures because they cannot be preserved natively in vitreous ice with traditional plunge freezing or, in some cases, even microfluidic assisted spray-plunge freezing. These recalcitrant specimens can touch the air-water interface, aggregate, or bind to the support film rather than remain suspended in the film's holes.

Cryogenic Electron Microscopy on Strongly Correlated Quantum Materials.

ConspectusQuantum materials refers to a class of materials with exotic properties that arise from the quantum mechanical nature of their constituent electrons, exhibiting, for example, high-temperature superconductivity, colossal magnetoresistivity, multiferroicity, and topological behavior. Quantum materials often have incompletely filled d- or f-electron shells with narrow energy bands, and the conduct of their electrons is strongly correlated. One distinct characteristic of the materials is that their electronic states are often spatially inhomogeneous and thus well suited for study using a spatially resolved electron beam with its great scattering power and sensitivity to atomic ionicity. Furthermore, most of these exotic properties only manifest at very low temperatures, posing a challenge to modern electron microscopy. It requires extraordinarily instrument stabilities at cryogenic temperatures with critical spatial, temporal, and energy resolutions in both static and dynamic manner to probe these materials. On the other hand, the ability to directly visualize the atomic, electronic and spin structures and inhomogeneities of quantum materials and correlate them to their functionalities creates enormous opportunities. At the most elementary levels of condensed matter physics, understanding the competing order of electron, spin, orbital, and lattice and their degrees of freedom, the impacts of defects and interfaces, and the site-specific quantum phenomena and phase transitions that give rise to the emergent behaviors allows us to discover and control novel materials for quantum information science and technologies.In this Account, several of our research examples are selected to highlight the use of cryogenic electron microscopy (cryo-EM) to study strongly correlated quantum materials. We focus on the critical roles of heterogeneity, interfaces, defects, and disorder in crystal structure, magnetic structure, and electronic structure to understand the physical properties of the materials that cryo-EM enables. We show how electron crystallography coupled with Bragg diffraction and diffuse scattering analysis empowers us to reveal the nature of structural modulations, lattice distortion, and phonons and how quantitative electron diffraction can be used to map the distributions of the valence electrons that bond atoms together. We exploit transformative advances in imaging capabilities including the use of femtosecond laser and ultrafast electron diffraction to probe electron-lattice interactions and photoinduced transitions beyond equilibrium of matter. We review our Lorentz phase microscopy studies to illustrate the intriguing transformations among various topological chiral spin states under applied magnetic field at various cryogenic temperatures. Finally, we show that atomically resolved imaging and electron energy-loss spectroscopy at 10 K can be used to understand interface-enhanced superconductivity. The wide range of research and progress on quantum materials at low temperature reported here may inspire and attract more researchers in this ever-expanding field of cryo-EM.

Validation, analysis and annotation of cryo-EM structures.

The process of turning 2D micrographs into 3D atomic models of the imaged macromolecules has been under rapid development and scrutiny in the field of cryo-EM. Here, some important methods for validation at several stages in this process are described. Firstly, how Fourier shell correlation of two independent maps and phase randomization beyond a certain frequency address the assessment of map resolution is reviewed. Techniques for local resolution estimation and map sharpening are also touched upon. The topic of validating models which are either built de novo or based on a known atomic structure fitted into a cryo-EM map is then approached. Map-model comparison using Q-scores and Fourier shell correlation plots is used to assure the agreement of the model with the observed map density. The importance of annotating the model with B factors to account for the resolvability of individual atoms in the map is illustrated. Finally, the timely topic of detecting and validating water molecules and metal ions in maps that have surpassed ∼2 Å resolution is described.

Retrospect and Prospect of Single Particle Cryo-Electron Microscopy: The Class of Integral Membrane Proteins as an Example.

A giant technological leap in the field of cryo-electron microscopy (cryo-EM) has assured the achievement of near-atomic resolution structures of biological macromolecules. As a recognition of this accomplishment, the Nobel Prize in Chemistry was awarded in 2017 to Jacques Dubochet, Joachim Frank, and Richard Henderson, the pioneers in the field of cryo-EM. Currently, the technique has become the method of choice for structural analysis of heterogeneous and intrinsically dynamic biological complexes. In the past few years, the most prolific branch of cryo-EM, single particle analysis, has revolutionized the structural biology field and structural investigation of membrane proteins, in particular. To achieve high-resolution structures of macromolecules in noncrystalline specimens, from sample and grid preparation to image acquisition, image data processing, and analysis of 3D maps, methodological advances in each of the steps play critical roles. In this Review, we discuss two areas in single particle cryo-EM, the remarkable developments in sample preparation strategies, particularly for membrane proteins, and breakthroughs in methodologies for molecular model building on the high-resolution 3D density maps that brought promises to resolve high-resolution (<4 Å) structures of biological macromolecules.

Current outcomes when optimizing 'standard' sample preparation for single-particle cryo-EM.

Although high-resolution single-particle cryo-electron microscopy (cryo-EM) is now producing a rapid stream of breakthroughs in structural biology, it nevertheless remains the case that the preparation of suitable frozen-hydrated samples on electron microscopy grids is often quite challenging. Purified samples that are intact and structurally homogeneous - while still in the test tube - may not necessarily survive the standard methods of making extremely thin, aqueous films on grids. As a result, it is often necessary to try a variety of experimental conditions before finally finding an approach that is optimal for the specimen at hand. Here, we summarize some of our collective experiences to date in optimizing sample preparation, in the hope that doing so will be useful to others, especially those new to the field. We also hope that an open discussion of these common challenges will encourage the development of more generally applicable methodology. Our collective experiences span a diverse range of biochemical samples and most of the commonly used variations in how grids are currently prepared. Unfortunately, none of the currently used optimization methods can be said, in advance, to be the one that ultimately will work when a project first begins. Nevertheless, there are some preferred first steps to explore when facing specific problems that can be more generally recommended, based on our experience and that of many others in the cryo-EM field.

DeepRes: a new deep-learning- and aspect-based local resolution method for electron-microscopy maps.

In this article, a method is presented to estimate a new local quality measure for 3D cryoEM maps that adopts the form of a 'local resolution' type of information. The algorithm (DeepRes) is based on deep-learning 3D feature detection. DeepRes is fully automatic and parameter-free, and avoids the issues of most current methods, such as their insensitivity to enhancements owing to B-factor sharpening (unless the 3D mask is changed), among others, which is an issue that has been virtually neglected in the cryoEM field until now. In this way, DeepRes can be applied to any map, detecting subtle changes in local quality after applying enhancement processes such as isotropic filters or substantially more complex procedures, such as model-based local sharpening, non-model-based methods or denoising, that may be very difficult to follow using current methods. It performs as a human observer expects. The comparison with traditional local resolution indicators is also addressed.

Electron cryo-microscopy for elucidating the dynamic nature of live-protein complexes

BackgroundRecent innovations in the field of electron cryo-microscopy (cryo-EM) have achieved structure determination of target macromolecules at “near-atomic resolution”. In addition, cryo-EM has the potential to deal with proteins in multiple conformations in close to physiological conditions. Scope of reviewThis is an overview of the latest technical and methodological developments in cryo-EM, especially key features for elucidating the dynamic nature of specimens. Major conclusionsIt is now possible to elucidate “near-atomic resolution” structures by selecting “well-behaved” particles in Single Particle Analysis (SPA) of cryo-EM images. General significanceCurrently, cryo-EM is utilized actively as an alternative to X-ray crystallography to obtain high-resolution structures of macromolecules, especially those which are hard to crystalize. One possible reason for difficulty in crystallization could be the dynamic nature of the specimen. New developments in the field of cryo-EM will make it possible to deal with specimen heterogeneity and reveal the dynamic nature of specimens.

Hands on Methods for High Resolution Cryo-Electron Microscopy Structures of Heterogeneous Macromolecular Complexes.

Electron microscopy of frozen hydrated samples (cryo-EM) is a powerful structural technique that allows the direct study of functional macromolecular complexes in an almost physiological environment. Protein macromolecular complexes are dynamic structures that usually hold together by an intricate network of protein-protein interactions that can be weak and transient. Moreover, a standard feature of many of these complexes is that they behave as nanomachines able to undergo functionally relevant conformational changes in one or several complex components. Among all the other main structural biology techniques, only cryo-EM has the potential of successfully dealing at the same time with both sample heterogeneity and inherent flexibility. The cryo-EM field is currently undergoing a revolution thanks to groundbreaking technical developments that have brought within our reach the possibility of solving the structure of biological complexes at atomic resolution. These technical developments have been mostly focused on new direct electron detector technology and improved sample preparation methods leading to better image quality. This fact has in turn required the development of new and better image processing algorithms to make the most of the higher quality data. The aim of this review is to provide a brief overview of some reported examples of single particle analysis strategies designed to find different conformational and compositional states within target macromolecular complex and specifically to deal with it to reach higher resolution information. Different image processing methodologies specifically aimed to symmetric or pseudo-symmetric protein complexes will also be discussed.

Biological Applications at the Cutting Edge of Cryo-Electron Microscopy.

Cryo-electron microscopy (cryo-EM) is a powerful tool for macromolecular to near-atomic resolution structure determination in the biological sciences. The specimen is maintained in a near-native environment within a thin film of vitreous ice and imaged in a transmission electron microscope. The images can then be processed by a number of computational methods to produce three-dimensional information. Recent advances in sample preparation, imaging, and data processing have led to tremendous growth in the field of cryo-EM by providing higher resolution structures and the ability to investigate macromolecules within the context of the cell. Here, we review developments in sample preparation methods and substrates, detectors, phase plates, and cryo-correlative light and electron microscopy that have contributed to this expansion. We also have included specific biological applications.

Applying a Modified Wavelet Shrinkage Filter to Improve Cryo-Electron Microscopy Imaging.

Cryo-electron microscopy (Cryo-EM) imaging has the unique potential to bridge the gap between cellular and molecular biology by revealing the structures of large macromolecular assemblies and cellular complexes. Therefore, cryo-EM three-dimensional (3D) reconstruction has been rapidly developed in recent several years and applied widely in life science research; however, it suffers from reduced contrast and low signal-to-noise ratios with a high degree of noise under low electron dose conditions, resulting in failures of many conventional filters. In this article, we explored a modified wavelet shrinkage filter (with optimal wavelet parameters: three-level decomposition, level-1 zeroed out, subband-dependent threshold, soft thresholding, and spline-based discrete dyadic wavelet transform) and extended its application in the cryo-EM field in two aspects: single-particle analysis and cryo-electron tomography. Its performance was assessed with simulation data and real cryo-EM experimental data. Compared with the undenoised results and conventional denoising techniques (e.g., Gaussian, median, and bilateral filters), the modified wavelet shrinkage filter maintained the resolution and contrast but reduced the noise, leading to higher quality images and more accurate measures of the biological structure. We expect that our study can provide benefits to cryo-EM applications: 3D reconstruction, visualization, structural analysis, and interpretation. All these data and programs are available.

An Open-Source Storage Solution for Cryo-Electron Microscopy Samples.

Cryo-electron microscopy (cryo-EM) enables the study of biological structures in situ in great detail and to solve protein structures at Ångstrom level resolution. Due to recent advances in instrumentation and data processing, the field of cryo-EM is a rapidly growing. Access to facilities and national centers that house the state-of-the-art microscopes is limited due to the ever-rising demand, resulting in long wait times between sample preparation and data acquisition. To improve sample storage, we have developed a cryo-storage system with an efficient, high storage capacity that enables sample storage in a highly organized manner. This system is simple to use, cost-effective and easily adaptable for any type of grid storage box and dewar and any size cryo-EM laboratory.