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16 pages, 2787 KiB

Open AccessArticle

CryoEM Workflow Acceleration with Feret Signatures

by Pierre Nottelet, Peter Van Blerkom, Xiao-Ping Xu, Dorit Hanein and Niels Volkmann

Int. J. Mol. Sci. 2024, 25(14), 7593; https://doi.org/10.3390/ijms25147593 - 11 Jul 2024

Cited by 1 | Viewed by 1418

Abstract

Common challenges in cryogenic electron microscopy, such as orientation bias, conformational diversity, and 3D misclassification, complicate single particle analysis and lead to significant resource expenditure. We previously introduced an in silico method using the maximum Feret diameter distribution, the Feret signature, to characterize [...] Read more.

Common challenges in cryogenic electron microscopy, such as orientation bias, conformational diversity, and 3D misclassification, complicate single particle analysis and lead to significant resource expenditure. We previously introduced an in silico method using the maximum Feret diameter distribution, the Feret signature, to characterize sample heterogeneity of disc-shaped samples. Here, we expanded the Feret signature methodology to identify preferred orientations of samples containing arbitrary shapes with only about 1000 particles required. This method enables real-time adjustments of data acquisition parameters for optimizing data collection strategies or aiding in decisions to discontinue ineffective imaging sessions. Beyond detecting preferred orientations, the Feret signature approach can serve as an early-warning system for inconsistencies in classification during initial image processing steps, a capability that allows for strategic adjustments in data processing. These features establish the Feret signature as a valuable auxiliary tool in the context of single particle analysis, significantly accelerating the structure determination process. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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11 pages, 5625 KiB

Open AccessArticle

The GoldX Fiducial Eraser

by Peter Van Blerkom, Armel Bezault, Cécile Sauvanet, Dorit Hanein and Niels Volkmann

Int. J. Mol. Sci. 2024, 25(13), 7442; https://doi.org/10.3390/ijms25137442 - 6 Jul 2024

Viewed by 1551

Abstract

Gold nanoparticles with sizes in the range of 5–15 nm are a standard method of providing fiducial markers to assist with alignment during reconstruction in cryogenic electron tomography. However, due to their high electron density and resulting contrast when compared to standard cellular [...] Read more.

Gold nanoparticles with sizes in the range of 5–15 nm are a standard method of providing fiducial markers to assist with alignment during reconstruction in cryogenic electron tomography. However, due to their high electron density and resulting contrast when compared to standard cellular or biological samples, they introduce artifacts such as streaking in the reconstructed tomograms. Here, we demonstrate a tool that automatically detects these nanoparticles and suppresses them by replacing them with a local background as a post-processing step, providing a cleaner tomogram without removing any sample relevant information or introducing new artifacts or edge effects from uniform density replacements. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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10 pages, 4425 KiB

Open AccessArticle

Mitigating the Blurring Effect of CryoEM Averaging on a Flexible and Highly Symmetric Protein Complex through Sub-Particle Reconstruction

by Diana S. Suder and Shane Gonen

Int. J. Mol. Sci. 2024, 25(11), 5665; https://doi.org/10.3390/ijms25115665 - 23 May 2024

Cited by 1 | Viewed by 1869

Abstract

Many macromolecules are inherently flexible as a feature of their structure and function. During single-particle CryoEM processing, flexible protein regions can be detrimental to high-resolution reconstruction as signals from thousands of particles are averaged together. This “blurring” effect can be difficult to overcome [...] Read more.

Many macromolecules are inherently flexible as a feature of their structure and function. During single-particle CryoEM processing, flexible protein regions can be detrimental to high-resolution reconstruction as signals from thousands of particles are averaged together. This “blurring” effect can be difficult to overcome and is possibly more pronounced when averaging highly symmetric complexes. Approaches to mitigating flexibility during CryoEM processing are becoming increasingly critical as the technique advances and is applied to more dynamic proteins and complexes. Here, we detail the use of sub-particle averaging and signal subtraction techniques to precisely target and resolve flexible DARPin protein attachments on a designed tetrahedrally symmetric protein scaffold called DARP14. Particles are first aligned as full complexes, and then the symmetry is reduced by alignment and focused refinement of the constituent subunits. The final reconstructions we obtained were vastly improved over the fully symmetric reconstructions, with observable secondary structure and side-chain placement. Additionally, we were also able to reconstruct the core region of the scaffold to 2.7 Å. The data processing protocol outlined here is applicable to other dynamic and symmetric protein complexes, and our improved maps could allow for new structure-guided variant designs of DARP14. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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19 pages, 9489 KiB

Open AccessArticle

Missing Wedge Completion via Unsupervised Learning with Coordinate Networks

by Dave Van Veen, Jesús G. Galaz-Montoya, Liyue Shen, Philip Baldwin, Akshay S. Chaudhari, Dmitry Lyumkis, Michael F. Schmid, Wah Chiu and John Pauly

Int. J. Mol. Sci. 2024, 25(10), 5473; https://doi.org/10.3390/ijms25105473 - 17 May 2024

Cited by 2 | Viewed by 2339

Abstract

Cryogenic electron tomography (cryoET) is a powerful tool in structural biology, enabling detailed 3D imaging of biological specimens at a resolution of nanometers. Despite its potential, cryoET faces challenges such as the missing wedge problem, which limits reconstruction quality due to incomplete data [...] Read more.

Cryogenic electron tomography (cryoET) is a powerful tool in structural biology, enabling detailed 3D imaging of biological specimens at a resolution of nanometers. Despite its potential, cryoET faces challenges such as the missing wedge problem, which limits reconstruction quality due to incomplete data collection angles. Recently, supervised deep learning methods leveraging convolutional neural networks (CNNs) have considerably addressed this issue; however, their pretraining requirements render them susceptible to inaccuracies and artifacts, particularly when representative training data is scarce. To overcome these limitations, we introduce a proof-of-concept unsupervised learning approach using coordinate networks (CNs) that optimizes network weights directly against input projections. This eliminates the need for pretraining, reducing reconstruction runtime by 3–20× compared to supervised methods. Our in silico results show improved shape completion and reduction of missing wedge artifacts, assessed through several voxel-based image quality metrics in real space and a novel directional Fourier Shell Correlation (FSC) metric. Our study illuminates benefits and considerations of both supervised and unsupervised approaches, guiding the development of improved reconstruction strategies. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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11 pages, 2260 KiB

Open AccessArticle

High-Resolution Cryo-Electron Microscopy Structure Determination of Haemophilus influenzae Tellurite-Resistance Protein A via 200 kV Transmission Electron Microscopy

by Nhi L. Tran, Skerdi Senko, Kyle W. Lucier, Ashlyn C. Farwell, Sabrina M. Silva, Phat V. Dip, Nicole Poweleit, Giovanna Scapin and Claudio Catalano

Int. J. Mol. Sci. 2024, 25(8), 4528; https://doi.org/10.3390/ijms25084528 - 20 Apr 2024

Viewed by 2600

Abstract

Membrane proteins constitute about 20% of the human proteome and play crucial roles in cellular functions. However, a complete understanding of their structure and function is limited by their hydrophobic nature, which poses significant challenges in purification and stabilization. Detergents, essential in the [...] Read more.

Membrane proteins constitute about 20% of the human proteome and play crucial roles in cellular functions. However, a complete understanding of their structure and function is limited by their hydrophobic nature, which poses significant challenges in purification and stabilization. Detergents, essential in the isolation process, risk destabilizing or altering the proteins’ native conformations, thus affecting stability and functionality. This study leverages single-particle cryo-electron microscopy to elucidate the structural nuances of membrane proteins, focusing on the SLAC1 bacterial homolog from Haemophilus influenzae (HiTehA) purified with diverse detergents, including n-dodecyl β-D-maltopyranoside (DDM), glycodiosgenin (GDN), β-D-octyl-glucoside (OG), and lauryl maltose neopentyl glycol (LMNG). This research not only contributes to the understanding of membrane protein structures but also addresses detergent effects on protein purification. By showcasing that the overall structural integrity of the channel is preserved, our study underscores the intricate interplay between proteins and detergents, offering insightful implications for drug design and membrane biology. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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22 pages, 10134 KiB

Open AccessArticle

Cryo-EM Map Anisotropy Can Be Attenuated by Map Post-Processing and a New Method for Its Estimation

by Ruben Sanchez-Garcia, Guillaume Gaullier, Jose Manuel Cuadra-Troncoso and Javier Vargas

Int. J. Mol. Sci. 2024, 25(7), 3959; https://doi.org/10.3390/ijms25073959 - 2 Apr 2024

Cited by 1 | Viewed by 1938

Abstract

One of the most important challenges in cryogenic electron microscopy (cryo-EM) is the substantial number of samples that exhibit preferred orientations, which leads to an uneven coverage of the projection sphere. As a result, the overall quality of the reconstructed maps can be [...] Read more.

One of the most important challenges in cryogenic electron microscopy (cryo-EM) is the substantial number of samples that exhibit preferred orientations, which leads to an uneven coverage of the projection sphere. As a result, the overall quality of the reconstructed maps can be severely affected, as manifested by the presence of anisotropy in the map resolution. Several methods have been proposed to measure the directional resolution of maps in tandem with experimental protocols to address the problem of preferential orientations in cryo-EM. Following these works, in this manuscript we identified one potential limitation that may affect most of the existing methods and we proposed an alternative approach to evaluate the presence of preferential orientations in cryo-EM reconstructions. In addition, we also showed that some of the most recently proposed cryo-EM map post-processing algorithms can attenuate map anisotropy, thus offering alternative visualization opportunities for cases affected by moderate levels of preferential orientations. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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20 pages, 5263 KiB

Open AccessArticle

Analysis of the Conformational Landscape of the N-Domains of the AAA ATPase p97: Disentangling the Continuous Conformational Variability in Partially Symmetrical Complexes

by Sepideh Valimehr, Rémi Vuillemot, Mohsen Kazemi, Slavica Jonic and Isabelle Rouiller

Int. J. Mol. Sci. 2024, 25(6), 3371; https://doi.org/10.3390/ijms25063371 - 16 Mar 2024

Viewed by 1989

Abstract

Single-particle cryo-electron microscopy (cryo-EM) has been shown to be effective in defining the structure of macromolecules, including protein complexes. Complexes adopt different conformations and compositions to perform their biological functions. In cryo-EM, the protein complexes are observed in solution, enabling the recording of [...] Read more.

Single-particle cryo-electron microscopy (cryo-EM) has been shown to be effective in defining the structure of macromolecules, including protein complexes. Complexes adopt different conformations and compositions to perform their biological functions. In cryo-EM, the protein complexes are observed in solution, enabling the recording of images of the protein in multiple conformations. Various methods exist for capturing the conformational variability through analysis of cryo-EM data. Here, we analyzed the conformational variability in the hexameric AAA + ATPase p97, a complex with a six-fold rotational symmetric core surrounded by six flexible N-domains. We compared the performance of discrete classification methods with our recently developed method, MDSPACE, which uses 3D-to-2D flexible fitting of an atomic structure to images based on molecular dynamics (MD) simulations. Our analysis detected a novel conformation adopted by approximately 2% of the particles in the dataset and determined that the N-domains of p97 sway by up to 60° around a central position. This study demonstrates the application of MDSPACE in analyzing the continuous conformational changes in partially symmetrical protein complexes, systems notoriously difficult to analyze due to the alignment errors caused by their partial symmetry. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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16 pages, 6087 KiB

Open AccessArticle

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

by Rémi Vuillemot, Mohamad Harastani, Ilyes Hamitouche and Slavica Jonic

Int. J. Mol. Sci. 2024, 25(1), 20; https://doi.org/10.3390/ijms25010020 - 19 Dec 2023

Viewed by 2225

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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15 pages, 27637 KiB

Open AccessArticle

Untangling Irregular Actin Cytoskeleton Architectures in Tomograms of the Cell with Struwwel Tracer

by Salim Sazzed, Peter Scheible, Jing He and Willy Wriggers

Int. J. Mol. Sci. 2023, 24(24), 17183; https://doi.org/10.3390/ijms242417183 - 6 Dec 2023

Cited by 1 | Viewed by 1725

Abstract

In this work, we established, validated, and optimized a novel computational framework for tracing arbitrarily oriented actin filaments in cryo-electron tomography maps. Our approach was designed for highly complex intracellular architectures in which a long-range cytoskeleton network extends throughout the cell bodies and [...] Read more.

In this work, we established, validated, and optimized a novel computational framework for tracing arbitrarily oriented actin filaments in cryo-electron tomography maps. Our approach was designed for highly complex intracellular architectures in which a long-range cytoskeleton network extends throughout the cell bodies and protrusions. The irregular organization of the actin network, as well as cryo-electron-tomography-specific noise, missing wedge artifacts, and map dimensions call for a specialized implementation that is both robust and efficient. Our proposed solution, Struwwel Tracer, accumulates densities along paths of a specific length in various directions, starting from locally determined seed points. The highest-density paths originating from the seed points form short linear candidate filament segments, which are further scrutinized and classified by users via inspection of a novel pruning map, which visualizes the likelihood of being a part of longer filaments. The pruned linear candidate filament segments are then iteratively fused into continuous, longer, and curved filaments based on their relative orientations, gap spacings, and extendibility. When applied to the simulated phantom tomograms of a Dictyostelium discoideum filopodium under experimental conditions, Struwwel Tracer demonstrated high efficacy, with F1-scores ranging from 0.85 to 0.90, depending on the noise level. Furthermore, when applied to a previously untraced experimental tomogram of mouse fibroblast lamellipodia, the filaments predicted by Struwwel Tracer exhibited a good visual agreement with the experimental map. The Struwwel Tracer framework is highly time efficient and can complete the tracing process in just a few minutes. The source code is publicly available with version 3.2 of the free and open-source Situs software package. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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15 pages, 12467 KiB

Open AccessArticle

An Improved Method for Growing Primary Neurons on Electron Microscopy Grids Co-Cultured with Astrocytes

by Ishika Kumar, Anju Paudyal, Anna Kádková, Michelle Stewart, Jakob Balslev Sørensen and Julika Radecke

Int. J. Mol. Sci. 2023, 24(20), 15191; https://doi.org/10.3390/ijms242015191 - 14 Oct 2023

Viewed by 2520

Abstract

With the increasing popularity of cryo-electron tomography (cryo-ET) in recent years, the quest to establish a method for growing primary neurons directly on electron microscopy grids (EM grids) has been ongoing. Here we describe a straightforward way to establish a mature neuronal network [...] Read more.

With the increasing popularity of cryo-electron tomography (cryo-ET) in recent years, the quest to establish a method for growing primary neurons directly on electron microscopy grids (EM grids) has been ongoing. Here we describe a straightforward way to establish a mature neuronal network on EM grids, which includes formation of synaptic contacts. These synapses were thin enough to allow for direct visualization of small filaments such as SNARE proteins tethering the synaptic vesicle (SV) to the active zone plasma membrane on a Titan Krios without prior focused ion-beam milling. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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22 pages, 1115 KiB

Open AccessArticle

Scipion-EM-ProDy: A Graphical Interface for the ProDy Python Package within the Scipion Workflow Engine Enabling Integration of Databases, Simulations and Cryo-Electron Microscopy Image Processing

by James M. Krieger, Carlos Oscar S. Sorzano and Jose Maria Carazo

Int. J. Mol. Sci. 2023, 24(18), 14245; https://doi.org/10.3390/ijms241814245 - 18 Sep 2023

Cited by 4 | Viewed by 2171

Abstract

Macromolecular assemblies, such as protein complexes, undergo continuous structural dynamics, including global reconfigurations critical for their function. Two fast analytical methods are widely used to study these global dynamics, namely elastic network model normal mode analysis and principal component analysis of ensembles of [...] Read more.

Macromolecular assemblies, such as protein complexes, undergo continuous structural dynamics, including global reconfigurations critical for their function. Two fast analytical methods are widely used to study these global dynamics, namely elastic network model normal mode analysis and principal component analysis of ensembles of structures. These approaches have found wide use in various computational studies, driving the development of complex pipelines in several software packages. One common theme has been conformational sampling through hybrid simulations incorporating all-atom molecular dynamics and global modes of motion. However, wide functionality is only available for experienced programmers with limited capabilities for other users. We have, therefore, integrated one popular and extensively developed software for such analyses, the ProDy Python application programming interface, into the Scipion workflow engine. This enables a wider range of users to access a complete range of macromolecular dynamics pipelines beyond the core functionalities available in its command-line applications and the normal mode wizard in VMD. The new protocols and pipelines can be further expanded and integrated into larger workflows, together with other software packages for cryo-electron microscopy image analysis and molecular simulations. We present the resulting plugin, Scipion-EM-ProDy, in detail, highlighting the rich functionality made available by its development. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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19 pages, 3192 KiB

Open AccessArticle

Extensive Angular Sampling Enables the Sensitive Localization of Macromolecules in Electron Tomograms

by Marten L. Chaillet, Gijs van der Schot, Ilja Gubins, Sander Roet, Remco C. Veltkamp and Friedrich Förster

Int. J. Mol. Sci. 2023, 24(17), 13375; https://doi.org/10.3390/ijms241713375 - 29 Aug 2023

Cited by 30 | Viewed by 4681

Abstract

Cryo-electron tomography provides 3D images of macromolecules in their cellular context. To detect macromolecules in tomograms, template matching (TM) is often used, which uses 3D models that are often reliable for substantial parts of the macromolecules. However, the extent of rotational searches in [...] Read more.

Cryo-electron tomography provides 3D images of macromolecules in their cellular context. To detect macromolecules in tomograms, template matching (TM) is often used, which uses 3D models that are often reliable for substantial parts of the macromolecules. However, the extent of rotational searches in particle detection has not been investigated due to computational limitations. Here, we provide a GPU implementation of TM as part of the PyTOM software package, which drastically speeds up the orientational search and allows for sampling beyond the Crowther criterion within a feasible timeframe. We quantify the improvements in sensitivity and false-discovery rate for the examples of ribosome identification and detection. Sampling at the Crowther criterion, which was effectively impossible with CPU implementations due to the extensive computation times, allows for automated extraction with high sensitivity. Consequently, we also show that an extensive angular sample renders 3D TM sensitive to the local alignment of tilt series and damage induced by focused ion beam milling. With this new release of PyTOM, we focused on integration with other software packages that support more refined subtomogram-averaging workflows. The automated classification of ribosomes by TM with appropriate angular sampling on locally corrected tomograms has a sufficiently low false-discovery rate, allowing for it to be directly used for high-resolution averaging and adequate sensitivity to reveal polysome organization. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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Review

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25 pages, 3435 KiB

Open AccessReview

Overview of Membrane Protein Sample Preparation for Single-Particle Cryo-Electron Microscopy Analysis

by Catherine Vénien-Bryan and Carlos A. H. Fernandes

Int. J. Mol. Sci. 2023, 24(19), 14785; https://doi.org/10.3390/ijms241914785 - 30 Sep 2023

Cited by 13 | Viewed by 5527

Abstract

Single-particle cryo-electron microscopy (cryo-EM SPA) has recently emerged as an exceptionally well-suited technique for determining the structure of membrane proteins (MPs). Indeed, in recent years, huge increase in the number of MPs solved via cryo-EM SPA at a resolution better than 3.0 Å [...] Read more.

Single-particle cryo-electron microscopy (cryo-EM SPA) has recently emerged as an exceptionally well-suited technique for determining the structure of membrane proteins (MPs). Indeed, in recent years, huge increase in the number of MPs solved via cryo-EM SPA at a resolution better than 3.0 Å in the Protein Data Bank (PDB) has been observed. However, sample preparation remains a significant challenge in the field. Here, we evaluated the MPs solved using cryo-EM SPA deposited in the PDB in the last two years at a resolution below 3.0 Å. The most critical parameters for sample preparation are as follows: (i) the surfactant used for protein extraction from the membrane, (ii) the surfactant, amphiphiles, nanodiscs or other molecules present in the vitrification step, (iii) the vitrification method employed, and (iv) the type of grids used. The aim is not to provide a definitive answer on the optimal sample conditions for cryo-EM SPA of MPs but rather assess the current trends in the MP structural biology community towards obtaining high-resolution cryo-EM structures. Full article

(This article belongs to the Special Issue Cryogenic Biomolecular Imaging and Image Analysis Techniques: Present and Future)

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