Scipion3: A workflow engine for cryo-electron microscopy image processing and structural biology.

Image-processing pipelines require the design of complex workflows combining many different steps that bring the raw acquired data to a final result with biological meaning. In the image-processing domain of cryo-electron microscopy single-particle analysis (cryo-EM SPA), hundreds of steps must be performed to obtain the three-dimensional structure of a biological macromolecule by integrating data spread over thousands of micrographs containing millions of copies of allegedly the same macromolecule. The execution of such complicated workflows demands a specific tool to keep track of all these steps performed.

Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs.

DNA regulation, replication and repair are processes fundamental to all known organisms and the sliding clamp proliferating cell nuclear antigen (PCNA) is central to all these processes. S-phase delaying protein 1 (Spd1) from S. pombe, an intrinsically disordered protein that causes checkpoint activation by inhibiting the enzyme ribonucleotide reductase, has one of the most divergent PCNA binding motifs known.

A new algorithm for particle weighted subtraction to decrease signals from unwanted components in single particle analysis.

Single particle analysis (SPA) in cryo-electron microscopy (cryo-EM) is highly used to obtain the near-atomic structure of biological macromolecules. The current methods allow users to produce high-resolution maps from many samples. However, there are still challenging cases that require extra processing to obtain high resolution.

Image processing tools for the validation of CryoEM maps.

The number of maps deposited in public databases (Electron Microscopy Data Bank, EMDB) determined by cryo-electron microscopy has quickly grown in recent years. With this rapid growth, it is critical to guarantee their quality. So far, map validation has primarily focused on the agreement between maps and models.

Development of a Microgram Scale Video-Microscopic Method to Investigate Dissolution Behavior of Poorly Water-Soluble Drugs.

Poor aqueous solubility is a common characteristic of new drug candidates, which leads to low or inconsistent oral bioavailability. This has sparked an interest in material efficient testing of solubility and dissolution rate. The aim was to develop a microgram scale video-microscopic method to screen the dissolution rates of poorly water-soluble drugs.

ScipionTomo: Towards cryo-electron tomography software integration, reproducibility, and validation.

Image processing in cryogenic electron tomography (cryoET) is currently at a similar state as Single Particle Analysis (SPA) in cryogenic electron microscopy (cryoEM) was a few years ago. Its data processing workflows are far from being well defined and the user experience is still not smooth. Moreover, file formats of different software packages and their associated metadata are not standardized, mainly since different packages are developed by different groups, focusing on different steps of the data processing pipeline.

On bias, variance, overfitting, gold standard and consensus in single-particle analysis by cryo-electron microscopy.

Cryo-electron microscopy (cryoEM) has become a well established technique to elucidate the 3D structures of biological macromolecules. Projection images from thousands of macromolecules that are assumed to be structurally identical are combined into a single 3D map representing the Coulomb potential of the macromolecule under study. This article discusses possible caveats along the image-processing path and how to avoid them to obtain a reliable 3D structure.

Advances in Xmipp for Cryo-Electron Microscopy: From Xmipp to Scipion.

Xmipp is an open-source software package consisting of multiple programs for processing data originating from electron microscopy and electron tomography, designed and managed by the Biocomputing Unit of the Spanish National Center for Biotechnology, although with contributions from many other developers over the world. During its 25 years of existence, Xmipp underwent multiple changes and updates. While there were many publications related to new programs and functionality added to Xmipp, there is no single publication on the Xmipp as a package since 2013.

Cryo-EM density maps adjustment for subtraction, consensus and sharpening.

Electron cryomicroscopy (cryo-EM) has emerged as a powerful structural biology instrument to solve near-atomic three-dimensional structures. Despite the fast growth in the number of density maps generated from cryo-EM data, comparison tools among these reconstructions are still lacking. Current proposals to compare cryo-EM data derived volumes perform map subtraction based on adjustment of each volume grey level to the same scale.

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.

Image Processing in Cryo-Electron Microscopy of Single Particles: The Power of Combining Methods.

Cryo-electron microscopy has established as a mature structural biology technique to elucidate the three-dimensional structure of biological macromolecules. The Coulomb potential of the sample is imaged by an electron beam, and fast semi-conductor detectors produce movies of the sample under study. These movies have to be further processed by a whole pipeline of image-processing algorithms that produce the final structure of the macromolecule.

Near-atomic structure of an atadenovirus reveals a conserved capsid-binding motif and intergenera variations in cementing proteins.

Of five known adenovirus genera, high-resolution structures are available only for mammalian-infecting mastadenoviruses. We present the first high-resolution structure of an adenovirus with nonmammalian host: lizard atadenovirus LAdV-2. We find a large conformational difference in the internal vertex protein IIIa between mast- and atadenoviruses, induced by the presence of an extended polypeptide.

Cryo-EM structure of enteric adenovirus HAdV-F41 highlights structural variations among human adenoviruses.

Enteric adenoviruses, one of the main causes of viral gastroenteritis in the world, must withstand the harsh conditions found in the gut. This requirement suggests that capsid stability must be different from that of other adenoviruses. We report the 4-Å-resolution structure of a human enteric adenovirus, HAdV-F41, and compare it with that of other adenoviruses with respiratory (HAdV-C5) and ocular (HAdV-D26) tropisms.

Algorithmic robustness to preferred orientations in single particle analysis by CryoEM.

The presence of preferred orientations in single particle analysis (SPA) by cryo-Electron Microscopy (cryoEM) is currently one of the hurdles preventing many structural analyses from yielding high-resolution structures. Although the existence of preferred orientations is mostly related to the grid preparation, in this technical note, we show that some image processing algorithms used for angular assignment and three-dimensional (3D) reconstruction are more robust than others to these detrimental conditions. We exemplify this argument with three different data sets in which the presence of preferred orientations hindered achieving a 3D reconstruction without artifacts or, even worse, a 3D reconstruction could never be achieved.

FSC-Q: a CryoEM map-to-atomic model quality validation based on the local Fourier shell correlation.

In recent years, advances in cryoEM have dramatically increased the resolution of reconstructions and, with it, the number of solved atomic models. It is widely accepted that the quality of cryoEM maps varies locally; therefore, the evaluation of the maps-derived structural models must be done locally as well. In this article, a method for the local analysis of the map-to-model fit is presented.

The Microbial Diversity of Non-Korean Kimchi as Revealed by Viable Counting and Metataxonomic Sequencing.

Kimchi is recognized worldwide as the flagship food of Korea. To date, most of the currently available microbiological studies on kimchi deal with Korean manufactures. Moreover, there is a lack of knowledge on the occurrence of eumycetes in kimchi.

Continuous flexibility analysis of SARS-CoV-2 spike prefusion structures.

Using a new consensus-based image-processing approach together with principal component analysis, the flexibility and conformational dynamics of the SARS-CoV-2 spike in the prefusion state have been analysed. These studies revealed concerted motions involving the receptor-binding domain (RBD), N-terminal domain, and subdomains 1 and 2 around the previously characterized 1-RBD-up state, which have been modeled as elastic deformations. It is shown that in this data set there are not well defined, stable spike conformations, but virtually a continuum of states.

Continuous flexibility analysis of SARS-CoV-2 Spike prefusion structures.

With the help of novel processing workflows and algorithms, we have obtained a better understanding of the flexibility and conformational dynamics of the SARS-CoV-2 spike in the prefusion state. We have re-analyzed previous cryo-EM data combining 3D clustering approaches with ways to explore a continuous flexibility space based on 3D Principal Component Analysis. These advanced analyses revealed a concerted motion involving the receptor-binding domain (RBD), N-terminal domain (NTD), and subdomain 1 and 2 (SD1 & SD2) around the previously characterized 1-RBD-up state, which have been modeled as elastic deformations.

Measurement of local resolution in electron tomography.

Resolution (global and local) is one of the most reported metrics of quality measurement in Single Particle Analysis (SPA). However, in electron tomography, the situation is different and its computation is not straightforward. Typically, resolution estimation is global and, therefore, reduces the assessment of a whole tomogram to a single number.

Integration of Cryo-EM Model Building Software in .

Advances in cryo-electron microscopy (cryo-EM) have made it possible to obtain structures of large biological macromolecules at near-atomic resolution. This "resolution revolution" has encouraged the use and development of modeling tools able to produce high-quality atomic models from cryo-EM density maps. Unfortunately, many practical problems appear when combining different packages in the same processing workflow, which make difficult the use of these tools by non-experts and, therefore, reduce their utility.

Re-examining the spectra of macromolecules. Current practice of spectral quasi B-factor flattening.

The analysis of structure factors in 3D cryo-EM Coulomb potential maps and their "enhancement" at the end of the reconstruction process is a well-established practice, normally referred to as sharpening. The aim is to increase contrast and, in this way, to help tracing the atomic model. The most common way to accomplish this enhancement is by means of the so-called B-factor correction, which applies a global filter to boost high frequencies with some dampening considerations related to noise amplification.

IDDomainSpotter: Compositional bias reveals domains in long disordered protein regions-Insights from transcription factors.

Protein domains constitute regions of distinct structural properties and molecular functions that are retained when removed from the rest of the protein. However, due to the lack of tertiary structure, the identification of domains has been largely neglected for long (>50 residues) intrinsically disordered regions. Here we present a sequence-based approach to assess and visualize domain organization in long intrinsically disordered regions based on compositional sequence biases.

Flexible workflows for on-the-fly electron-microscopy single-particle image processing using Scipion.

Electron microscopy of macromolecular structures is an approach that is in increasing demand in the field of structural biology. The automation of image acquisition has greatly increased the potential throughput of electron microscopy. Here, the focus is on the possibilities in Scipion to implement flexible and robust image-processing workflows that allow the electron-microscope operator and the user to monitor the quality of image acquisition, assessing very simple acquisition measures or obtaining a first estimate of the initial volume, or the data resolution and heterogeneity, without any need for programming skills.

Automatic local resolution-based sharpening of cryo-EM maps.

Motivation: Recent technological advances and computational developments have allowed the reconstruction of Cryo-Electron Microscopy (cryo-EM) maps at near-atomic resolution. On a typical workflow and once the cryo-EM map has been calculated, a sharpening process is usually performed to enhance map visualization, a step that has proven very important in the key task of structural modeling. However, sharpening approaches, in general, neglects the local quality of the map, which is clearly suboptimal.

The PCNA interaction motifs revisited: thinking outside the PIP-box.

Proliferating cell nuclear antigen (PCNA) is a cellular hub in DNA metabolism and a potential drug target. Its binding partners carry a short linear motif (SLiM) known as the PCNA-interacting protein-box (PIP-box), but sequence-divergent motifs have been reported to bind to the same binding pocket. To investigate how PCNA accommodates motif diversity, we assembled a set of 77 experimentally confirmed PCNA-binding proteins and analyzed features underlying their binding affinity.