Cryo-EM and Single-Particle Analysis with Scipion

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.

Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) have transformed structural biology by enabling the visualization of biological macromolecules at near-atomic resolution. Since its establishment in 2002, the Electron Microscopy Data Bank (EMDB) has played a pivotal role in this “Resolution Revolution,” serving as a central repository for cryo-EM data. Over the past decade, major advances in detector technology, image processing, and analytical methodologies have led to a surge in cryo-EM structure depositions. This study provides a comprehensive analysis of temporal trends in the EMDB, highlighting the growing number of entries, improvements in resolution, and the evolution of detector usage. It emphasizes the rise of single-particle analysis (SPA) as the dominant cryo-EM technique and examines how high-resolution detectors have enhanced data quality. Additionally, it explores resolution standards across leading scientific journals, underscoring the role of EMDB not only as a data archive but also as a catalyst for innovation in structural biology.

Single particle analysis integrated with microscopy: A high-throughput approach for reconstructing icosahedral particles

A microCT imaging protocol for reproducible and efficient quantitative morphometric analysis (QMA) of joint structures of the in situ mouse tibio-femoral joint.

Recent major advancements in cryo-electron microscopy (cryo-EM) have enabled high-resolution structural analysis, accompanied by developments in image processing software packages for single-particle analysis (SPA). SPA facilitates the 3D reconstruction of proteins and macromolecular complexes from numerous individual particles. In this study, we systematically evaluated the impact of symmetry parameters and particle quantity on the 3D reconstruction efficiency using the dihydrolipoyl acetyltransferase (E2) inner core of the pyruvate dehydrogenase complex (PDC). We specifically examined how inappropriate symmetry constraints can introduce structural artifacts and distortions, underscoring the necessity for accurate symmetry determination through rigorous validation methods such as directional Fourier shell correlation (FSC) and local-resolution mapping. Additionally, our analysis demonstrates that efficient reconstructions can be achieved with a moderate particle number, significantly reducing computational costs without compromising structural accuracy. We further contextualize these results by discussing recent developments in SPA workflows and hardware optimization, highlighting their roles in enhancing reconstruction accuracy and computational efficiency. Overall, our comprehensive benchmarking provides strategic insights that will facilitate the optimization of SPA experiments, particularly in resource-limited settings, and offers practical guidelines for accurately determining symmetry and particle quantity during cryo-EM data processing.

Purpose The dicentric chromosome assay (DCA), recognized as the gold standard for biological dosimetry in emergency radiation exposure, involves multiple key steps. Among these, metaphase image selection remains ambiguous and particularly challenging for beginners. This study evaluates the impact of metaphase image selection on dicentric (Dic) frequency during initial training. Materials and methods Peripheral blood (PB) from three healthy donors was irradiated with 2 Gy X-rays and incubated at 37 °C for 2 h for DNA repair. PB samples were then cultured for 48 h with phytohemagglutinin and colcemid. After fixation, metaphase spreads were prepared, Giemsa-stained, and images were captured by specific microscope imaging and image processing software. Three beginners independently selected and analyzed 50 metaphase images each. A trainer with experience in performing radiation emergency related DCA then reviewed and removed unsuitable images. The beginners reanalyzed Dic frequencies, supplementing with additional images as needed to reach 50 metaphases. Dic frequencies were compared before and after image refinement. Results Dic frequencies were lower in metaphase image pools selected by beginners compared to those refined by the trainer. The novices’ selections included numerous over-condensed metaphases, making Dic detection difficult. Conclusion Blood culture for 48 h permanent colcemid treatment increases the occurrence of over-condensed chromosomes, affecting Dic scoring. This study highlights the importance of proper training in metaphase image selection and structured education on quality assessment, including the use of specific microscope imaging and image processing software to ensure accurate biological dosimetry.

The Electron Microscopy eXchange (EMX) initiative.

Single Particle Imaging (SPI) with intense coherent X-ray pulses from X-ray free-electron lasers (XFELs) has the potential to produce molecular structures without the need for crystallization or freezing. Here we present a dataset of 285,944 diffraction patterns from aerosolized Coliphage PR772 virus particles injected into the femtosecond X-ray pulses of the Linac Coherent Light Source (LCLS). Additional exposures with background information are also deposited. The diffraction data were collected at the Atomic, Molecular and Optical Science Instrument (AMO) of the LCLS in 4 experimental beam times during a period of four years. The photon energy was either 1.2 or 1.7 keV and the pulse energy was between 2 and 4 mJ in a focal spot of about 1.3 μm x 1.7 μm full width at half maximum (FWHM). The X-ray laser pulses captured the particles in random orientations. The data offer insight into aerosolised virus particles in the gas phase, contain information relevant to improving experimental parameters, and provide a basis for developing algorithms for image analysis and reconstruction.

Chromatic and achromatic (AC) assessments of camouflage textiles have been critical to the defense researchers for concealment, detection, recognition, and identification (CDRI) of target signature against multidimensional combat background (CB). AC assessment and camouflage measurement techniques are simulated and experimented for assessment of camouflage textiles against CB. This model has been demonstrated for color measurement spectrophotometer, scanning electron microscopy (SEM), digital imaging, hyperspectral imaging, and image processing software (ImageJ) for the advancement and establishment of AC camouflage textiles assessment. The chromatic variations of 48 artificial target objects (TOBs) have been synthesized by image processing; the technique can be implemented for defense CB-CDRI assessment. Microstructural variation versus optical signal of woodland, desertland and stoneland CB materials have been elucidated by SEM magnification. The achromatic variation of CB materials have been demonstrated for the replacement of optical signal against modern remote sensing device to the imaging sensor. Color difference (Δ E), microstructural variations, pixel variations to imaging signal and standard deviation of CB materials have been represented for remote sensing surveillance of defense applications against TOB-CB-CDRI. Technical simulation of color, texture, gloss, and pixel intensity has been derived for AC-CDRI assessment of camouflage textiles in TOBs-CB environment.

With contrast enhancement technique, cryo-transmission electron microscopy (cryo-TEM) can be more advantageous. A Zernike phase plate (ZPP) and a hole-free phase plate (HFPP) are reported to have capability to enhance contrast [1, 2, 3, 4]. The ZPP is made of thin amorphous carbon film with a center hole, and the HFPP is made of thinner carbon film with no hole. In this study, we report the results with these phase plates on cryo-microscopic techniques such as cryo-TEM tomography and single particle analysis. With ZPP and HFPP, images in a close-to-focus condition exhibited higher contrast compared to defocus phase-contrast images (see Fig. A-C). The image with HFPP shows reduced fringe contrast (Fig. C), compared to the image with ZPP (Fig. B). In addition, the HFPP does not require any alignment procedure due to lack of a center hole. Thus, the data acquisition with HFPP could be easier than that with ZPP. [1] R Danev and K Nagayama, Ultramicroscopy 88 (2001) 243-252. [2] M Malac et al, Ultramicroscopy 118 (2012), 77-89. [3] M Malac et al, U.S. Patent US 8,785,850. [4] M Malac et al, JP. Patent 2011-151019. Figure: Cryo-TEM images of ice-embedded GroEL particles acquired with a few microns of defocus (A), ZPP (B) and HFPP (C). The images with ZPP and HFPP were taken with a close-to-focus condition. C2-P-07 doi:10.1093/jmicro/dfv303

In living organisms, biological macromolecules are intrinsically flexible and naturally exist in multiple conformations. Modern electron microscopy, especially at liquid nitrogen temperatures (cryo-EM), is able to visualise biocomplexes in nearly native conditions and in multiple conformational states. The advances made during the last decade in electronic technology and software development have led to the revelation of structural variations in complexes and also improved the resolution of EM structures. Nowadays, structural studies based on single particle analysis (SPA) suggests several approaches for the separation of different conformational states and therefore disclosure of the mechanisms for functioning of complexes. The task of resolving different states requires the examination of large datasets, sophisticated programs, and significant computing power. Some methods are based on analysis of two-dimensional images, while others are based on three-dimensional studies. In this review, we describe the basic principles implemented in the various techniques that are currently used in the analysis of structural conformations and provide some examples of successful applications of these methods in structural studies of biologically significant complexes.

High-resolution structures of membrane and soluble proteins can be obtained by cryo-electron crystallography, given highly-ordered cryo-preparations of perfectly flat 2D crystals are available. Studies of membrane proteins, which are embedded into a lipid membrane, mimicking the native cell membrane, are of particular biological interest. However there are multiple reasons why electron crystallography is far from being a mainstream protein structure determination technique. In this thesis we address three major difficulties of electron crystallography: (i) resolution loss due to not perfectly flat crystals, (ii) reliable high-throughput automatic image processing and (iii) correction of electron beam-induced motion of the sample.
\n
\nThe conventional electron crystallography image processing procedure assumes perfectly flat 2D crystals, which are almost impossible to obtain. Our new processing approach, described in Chapter 2, weakens this assumption tremendously. Traditional processing assigns the same tilt geometry to all proteins of one 2D crystal. Thus local tilt geometry variations, due to not perfectly flat crystals, are neglected. We developed an algorithm that optimizes the tilt geometry of each protein separately, while exploiting the correlation between neighboring proteins. The new method proves the feasibility of this approach, improves the achieved resolution and opens the doors to new studies, i.e. structural studies of membrane proteins embedded into lipid vesicles.
\n
\nRecently a new generation of digital detectors tremendously changed the cryo-electron microscopy field. Beside a significantly increased signal-to-noise ratio these detectors record dose-fractionated movies of the sample under the electron beam. Previous cameras only recorded one image instead. This new exposure mode enables the computational correction for beam-induced sample movements. In Chapter 3 we describe a new “real-time” automation pipeline for electron crystallography using direct electron detectors. The novel pipeline automatically corrects for homogeneous sample drift on frame level and processes the acquired images automatically. Both, the time-to-solution and the quality of the obtained 3D reconstructions are significantly improved.
\n
\nHeterogeneous beam-induced sample movements are the most severe resolution- limiting factor in modern cryo-electron crystallography. In Chapter 4 we present an algorithm, termed movie-mode unbending, which corrects for inhomogeneous beam- induced sample drift. In contrast to the previous homogeneous drift-correction, the novel algorithm can correct for locally varying beam-induced sample motion. This novel approach significantly increases the resolution of electron cryo-crystallographic studies recorded on the latest detectors.
\n
\nChapter 5 covers multiple successful applications of the methods introduced above. For instance the real-time drift-correction module of the developed automation pipeline was used for multiple near-atomic resolution single particle and helical image processing projects. The high-throughput crystal image processing was applied to different kinds of 2D crystals and enabled the qualitative assessment of different sample preparation methods. Additionally we describe the implementation of a high-throughput single particle automation pipeline, which will enable the generation of near-atomic resolution single particle cryo-electron microscopy density maps on a daily basis.

A long-sought goal in structural biology has been the imaging of membrane proteins in their membrane environments. This goal has been achieved with electron crystallography in those special cases where a protein forms highly ordered arrays in lipid bilayers. It has also been achieved by NMR methods in proteins up to 50 kilodaltons (kDa) in size, although milligram quantities of protein and isotopic labelling are required. For structural analysis of large soluble proteins in microgram quantities, an increasingly powerful method that does not require crystallization is single-particle reconstruction from electron microscopy of cryogenically cooled samples (electron cryomicroscopy (cryo-EM)). Here we report the first single-particle cryo-EM study of a membrane protein, the human large-conductance calcium- and voltage-activated potassium channel (BK), in a lipid environment. The new method is called random spherically constrained (RSC) single-particle reconstruction. BK channels, members of the six-transmembrane-segment (6TM) ion channel family, were reconstituted at low density into lipid vesicles (liposomes), and their function was verified by a potassium flux assay. Vesicles were also frozen in vitreous ice and imaged in an electron microscope. From images of 8,400 individual protein particles, a three-dimensional (3D) reconstruction of the BK channel and its membrane environment was obtained at a resolution of 1.7-2.0 nm. Not requiring the formation of crystals, the RSC approach promises to be useful in the structural study of many other membrane proteins as well.

Both single-particle analysis and electron tomography are special tools for the structural analysis of biological components by transmission electron microscopy. Single-particle analysis enables us to determine the structures of relatively large proteins and macromolecular complexes without crystallisation. Electron tomography is often used to visualise organelles and subcellular structures at molecular resolution. Recently, the scope of electron tomography has been widened due to the introduction of an automatic data collection system, cryo-electron microscopy, and improved sample preparation techniques. Application of electron tomography to single-particle analysis will be capable of analysing dynamics of large proteins. The techniques described in this review are expected to bridge the gap between the atomic resolution structures of protein molecules and cellular structures obtained by light microscopy.

In cryo-electron microscopy (cryo-EM), the Wiener filter is the optimal operation – in the least-squares sense – of merging a set of aligned low signal-to-noise ratio (SNR) micrographs to obtain a class average image with higher SNR. However, the condition for the optimal behavior of the Wiener filter is that the signal of interest shows stationary characteristic thoroughly, which cannot always be satisfied. In this paper, we propose substituting the conventional Wiener filter, which encompasses the whole image for denoising, with its local adaptive implementation, which denoises the signal locally. We compare our proposed local adaptive Wiener filter (LA-Wiener filter) with the conventional class averaging method using a simulated dataset and an experimental cryo-EM dataset. The visual and numerical analyses of the results indicate that LA-Wiener filter is superior to the conventional approach in single particle reconstruction (SPR) applications.