Scipion: a software framework toward integration, reproducibility and validation in 3D Electron Microscopy

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Escuela Politécnica Superior, Departamento de Ingeniería Informática. Fecha de lectura : 25-10-2017In the past few years, 3D electron microscopy (3DEM) has undergone a revolution in instrumentation and methodology. One of the central players in this wide-reaching change is the continuous development of image processing software. Here we present Scipion, a software framework for integrating several 3DEM software packages through a work owbased approach. Scipion allows the execution of reusable, standardized, traceable and reproducible image-processing protocols. These protocols incorporate tools from di erent programs while providing full interoperability among them. Scipion is an open-source project that can be downloaded from http://scipion.cnb.csic.es

Cryo-EM and the elucidation of new macromolecular structures: Random Conical Tilt revisited

Cryo-Electron Microscopy (cryo-EM) of macromolecular complexes is a fundamental structural biology technique which is expanding at a very fast pace. Key to its success in elucidating the three-dimensional structure of a macromolecular complex, especially of small and non-symmetric ones, is the ability to start from a low resolution map, which is subsequently refined with the actual images collected at the microscope. There are several methods to produce this first structure. Among them, Random Conical Tilt (RCT) plays a prominent role due to its unbiased nature (it can create an initial model based on experimental measurements). In this article, we revise the fundamental mathematical expressions supporting RCT, providing new expressions handling all key geometrical parameters without the need of intermediate operations, leading to improved automation and overall reliability, essential for the success of cryo-EM when analyzing new complexes. We show that the here proposed RCT workflow based on the new formulation performs very well in practical cases, requiring very few image pairs (as low as 13 image pairs in one of our examples) to obtain relevant 3D maps.We thank Dr. Llorca for his support during the acquisition of the C3b images and Dr. Shaikh for his support in the use of Spider for the RCT reconstructions. The authors would like to acknowledge economical support from the Spanish Ministry of Economy and Competitiveness through grants AIC-A-2011-0638 and BIO2013-44647-R, the Comunidad de Madrid through grant CAM (S2010/BMD-2305), as well as a postdoctoral Juan de la Cierva grant with reference JCI-2011-10185 to Javier Vargas. Vahid Abrishami is a holder of La Caixa scholarship and C.O.S. Sorzano is recipient of a Ramon y Cajal fellowship

Structure and dynamics of single-isoform recombinant Neuronal Human Tubulin

Microtubules are polymers that cycle stochastically between polymerization and depolymerization i.e., they exhibit 'dynamic instability'. This behavior is crucial for cell division, motility and differentiation. While studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogenous, isotypically pure, engineered tubulin. Here we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2 Å structure of unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but is distinguished by subtle differences at polymerization interfaces, which are hotspots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogenous microtubules undergo dynamic instability but they polymerize slower and catastrophe less frequently. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient, but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogenous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well-defined tubulin species and is a first and necessary step towards uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics

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MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function

Assessment of protein-protein interfaces in cryo-EM derived assemblies

Structures of macromolecular assemblies derived from cryo-EM maps often contain errors that become more abundant with decreasing resolution. Despite efforts in the cryo-EM community to develop metrics for map and atomistic model validation, thus far, no specific scoring metrics have been applied systematically to assess the interface between the assembly subunits. Here, we comprehensively assessed protein–protein interfaces in macromolecular assemblies derived by cryo-EM. To this end, we developed Protein Interface-score (PI-score), a density-independent machine learning-based metric, trained using the features of protein–protein interfaces in crystal structures. We evaluated 5873 interfaces in 1053 PDB-deposited cryo-EM models (including SARS-CoV-2 complexes), as well as the models submitted to CASP13 cryo-EM targets and the EM model challenge. We further inspected the interfaces associated with low-scores and found that some of those, especially in intermediate-to-low resolution (worse than 4 Å) structures, were not captured by density-based assessment scores. A combined score incorporating PI-score and fit-to-density score showed discriminatory power, allowing our method to provide a powerful complementary assessment tool for the ever-increasing number of complexes solved by cryo-EM

Hybrid Electron Microscopy Normal Mode Analysis graphical interface and protocol

International audienceThis article presents an integral graphical interface to the Hybrid Electron Microscopy Normal Mode Analysis (HEMNMA) approach that was developed for capturing continuous motions of large macromolecular complexes from single-particle EM images. HEMNMA was shown to be a good approach to analyze multiple conformations of a macromolecular complex but it could not be widely used in the EM field due to a lack of an integral interface. In particular, its use required switching among different software sources as well as selecting modes for image analysis was difficult without the graphical interface. The graphical interface was thus developed to simplify the practical use of HEMNMA. It is implemented in the open-source software package Xmipp 3.1 (http://xmipp.cnb.csic.es) and only a small part of it relies on MATLAB that is accessible through the main interface. Such integration provides the user with an easy way to perform the analysis of macromolecular dynamics and forms a direct connection to the single-particle reconstruction process. A step-by-step HEMNMA protocol with the graphical interface is given in full details in Supplementary material. The graphical interface will be useful to experimentalists who are interested in studies of continuous conformational changes of macromolecular complexes beyond the modeling of continuous heterogeneity in single particle reconstruction

Macromolecular Dynamics by Hybrid Electron Microscopy Normal Mode Analysis

International audienceStructural changes are critical for biological functions of proteins and describing conformational changes in large macromolecular complexes is a major challenge. To allow full studies of gradual conformational changes, we have recently developed Hybrid Electron Microscopy Normal Mode Analysis (HEMNMA) [1]. HEMNMA uses normal modes to elastically align electron microscopy (EM) images with a reference structure, in order to determine the conformations present in images and evaluate their pertinence. HEMNMA allows analyzing gradual changes more extensively than other EM methods, as HEMNMA gives full dynamics while other EM methods give only a few conformations. The computed conformational distribution allows modeling of transition pathways. Here, we show the performance of HEMNMA using simulated data (Fig. 1). The results of the method with experimental EM images will be shown at the symposium. Also, we will show a user-friendly graphical interface to the method that we have recently built within the Xmipp software. Figure 1 shows the potential of the method to analyze any gradual-type conformational change. The synthetic conformational changes were computed using a crystal structure of Tomato Bushy Stunt Virus (TBSV) [2]. A set of 4000 images (size: 128 2 pixels; pixel size: (3.2 Å) 2) was generated for random orientations and positions of the reference structure displaced along a linear combination of mode 28 (mainly related to the capsid expansion), mode 80 (mainly related to the movement of subunits away and towards the 5-fold symmetry axis) and mode 107 (mainly related to the rotation of subunits around the 5-fold symmetry axis). The contribution of mode 28 (deformation amplitude along the mode 28) was arbitrarily chosen to be distributed uniformly in the range [0,2000] and the contributions of the modes 80 and 107 were chosen to be linearly related to the contribution of the mode 28 (Fig. 1a). Two out-of-plane rotations were distributed uniformly in the ranges [0°,360°] and [0°,180°], the in-plane rotation was 0°, and the in-plane translations were distributed uniformly in the range [–5,5] pixels. Noise (SNR=0.05) and CTF (defocus of 2 μm, 200 kV microscope, spherical aberration of 0.5 mm) were applied onto the computed projections using the model with noise before and after the CTF [3]. Each synthetic image was aligned elastically with the reference structure to estimate five rigid-body parameters and three deformation amplitudes (for three used modes). We compared the results of principal component analysis (PCA) of the computed deformation amplitudes with the results of PCA of the ground-truth deformation amplitudes, using the first two principal axes (Fig. 1b-c). The mean RMSD of 0.54 Å was obtained along the linear-regression lines through the computed and ground-truth conformations (Fig. 1b-c). We also compared the structures computed by 3D reconstruction (Fig. 1d-g) from 7 images groups of similar sizes (Fig. 1b-c). The cross-correlation of 0.98-0.99 was obtained between the structures reconstructed for the ground-truth and computed alignments. The conformational change for both the ground-truth parameters (Fig. 1d for the sequence of classes 1-4; Fig. 1e for the sequence of classes 1-2'-3'-4') and the computed parameters (Fig. 1f for the sequence of classes 1-4; 121