Wavelets filtering for classification of very noisy electron microscopic single particles images- application on structure determination of VP5-VP19C recombinant

BACKGROUND: Images of frozen hydrated [vitrified] virus particles were taken close-to-focus in an electron microscope containing structural signals at high spatial frequencies. These images had very low contrast due to the high levels of noise present in the image. The low contrast made particle selection, classification and orientation determination very difficult. The final purpose of the classification is to improve the signal-to-noise ratio of the particle representing the class, which is usually the average. In this paper, the proposed method is based on wavelet filtering and multi-resolution processing for the classification and reconstruction of this very noisy data. A multivariate statistical analysis (MSA) is used for this classification. RESULTS: The MSA classification method is noise dependant. A set of 2600 projections from a 3D map of a herpes simplex virus -to which noise was added- was classified by MSA. The classification shows the power of wavelet filtering in enhancing the quality of class averages (used in 3D reconstruction) compared to Fourier band pass filtering. A 3D reconstruction of a recombinant virus (VP5-VP19C) is presented as an application of multi-resolution processing for classification and reconstruction. CONCLUSION: The wavelet filtering and multi-resolution processing method proposed in this paper offers a new way for processing very noisy images obtained from electron cryo-microscopes. The multi-resolution and filtering improves the speed and accuracy of classification, which is vital for the 3D reconstruction of biological objects. The VP5-VP19C recombinant virus reconstruction presented here is an example, which demonstrates the power of this method. Without this processing, it is not possible to get the correct 3D map of this virus

Orientation determination by wavelets matching for 3D reconstruction of very noisy electron microscopic virus images

BACKGROUND: In order to perform a 3D reconstruction of electron microscopic images of viruses, it is necessary to determine the orientation (Euler angels) of the 2D projections of the virus. The projections containing high resolution information are usually very noisy. This paper proposes a new method, based on weighted-projection matching in wavelet space for virus orientation determination. In order to speed the retrieval of the best match between projections from a model and real virus particle, a hierarchical correlation matching method is also proposed. RESULTS: A data set of 600 HSV-1 capsid particle images in different orientations was used to test the proposed method. An initial model of about 40 Å resolutions was used to generate projections of an HSV-1 capsid. Results show that a significant improvement, in terms of accuracy and speed, is obtained for the initial orientation estimates of noisy herpes virus images. For the bacteriophage (P22), the proposed method gave the correct reconstruction compared to the model, while the classical method failed to resolve the correct orientations of the smooth spherical P22 viruses. CONCLUSION: This paper introduces a new method for orientation determination of low contrast images and highly noisy virus particles. This method is based on weighted projection matching in wavelet space, which increases the accuracy of the orientations. A hierarchical implementation of this method increases the speed of orientation determination. The estimated number of particles needed for a higher resolution reconstruction increased exponentially. For a 6 Å resolution reconstruction of the HSV virus, 50,000 particles are necessary. The results show that the proposed method reduces the amount of data needed in a reconstruction by at least 50 %. This may result in savings 2 to 3 man-years invested in acquiring images from the microscope and data processing. Furthermore, the proposed method is able to determine orientations for some difficult particles like P22 with accuracy and consistency. Recently a low PH sindbis capsid was determined with the proposed method, where other methods based on the common line fail

Automatic particle detection in digitized electron micrographs

High resolution structural analysis of biological complexes can be carried out by single particle electron microscopy where a large number of particle images are available. Many approaches to automate the process of selection of particle positions from digitized electron micrograph images have been described, but so far none has proved as good as manual selection. This thesis describes a method which I have developed to locate such biological complexes by matching small boxed areas to a set of reference images using the radius of gyration, complemented by a series of other simple criteria. From the reference images, parameters such as the ratio between the average density of the central area and that in its surrounding band, and the density sum and variance are calculated. They are compared with corresponding values from a moving square window of densities extracted from the micrograph, and the coordinates of successfully matched candidate squares are recorded. Since the same particle is detected in a series of overlapping windows, candidates found to be within close proximity are grouped, and the best-fitting one is selected from each cluster. Along with a small stack of boxed reference images, a few specified parameter values, such as the particle radius and the minimum acceptable distance between particle centres are required to select the windows. Micrograph labels and other areas that do not contain appropriate specimens are automatically ignored in order to minimize false positives, and reduce the computing time. A computer program SLEUTH written to carry out this method of automatic particle detection includes a graphical user interface to assist the user in setting up the parameter values. The program has been tested successfully on a variety of different biological structures, from both negatively stained and ice-embedded specimens

Statistical Reconstruction Methods for 3D Imaging of Biological Samples with Electron Microscopy

Electron microscopy has emerged as the leading method for the in vivo study of biological structures such as cells, organelles, protein molecules and virus like particles. By providing 3D images up to near atomic resolution, it plays a significant role in analyzing complex organizations, understanding physiological functions and developing medicines. The 3D images representing the electrostatic potential distribution are reconstructed by utilizing the 2D projection images of the target acquired by electron microscope. There are two main 3D reconstruction techniques in the field of electron microscopy: electron tomography (ET) and single particle reconstruction (SPR). In ET, the projection images are acquired by rotating the specimen for different angles. In SPR, the projection images are obtained by analyzing the images of multiple objects representing the same structure. Then, the tomographic reconstruction methods are applied in both methods to obtain the 3D image through the 2D projections.Physical and mechanical limitations can prevent to acquire projection images that cover the projection angle space completely and uniformly. Incomplete and non-uniform sampling of the projection angles results in anisotropic resolution in the image plane and generates artifacts. Another problem is that the total applied dose of electrons is limited in order to prevent the radiation damage to the biological target. Therefore, limited number of projection images with low signal to noise ratio can be used in the reconstruction process. This affects the resolution of the reconstructed image significantly. This study presents statistical methods to overcome these major challenges to obtain precise and high resolution images in electron microscopy.Statistical image reconstruction methods have been successful in recovering a signal from imperfect measurements due to their capability of utilizing a priori information. First, we developed a sequential application of a statistical method for ET. Then we extended the method to support projection angles freely distributed in 3D space and applied the method in SPR. In both applications, we observed the strength of the method in projection gap filling, robustness against noise, and resolving the high resolution details in comparison with the conventional reconstruction methods. Afterwards, we improved the method in terms of computation time by incorporating multiresolution reconstruction. Furthermore, we developed an adaptive regularization method to minimize the parameters required to be set by the user. We also proposed the local adaptive Wiener filter for the class averaging step of SPR to improve the averaging accuracy.The qualitative and quantitative analysis of the reconstructions with phantom and experimental datasets has demonstrated that the proposed reconstruction methods outperform the conventional reconstruction methods. These statistical approaches provided better image accuracy and higher resolution compared with the conventional algebraic and transfer domain based reconstruction methods. The methods provided in this study contribute to enhance our understanding of cellular and molecular structures by providing 3D images of those with improved accuracy and resolution

Local quality assessment of cryo-EM reconstructions and its applications.

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física de Materiales. Fecha de lectura: 28-05-2019The cryo-Electron Microscopy (cryoem) addressed to the elucidation of macromolecular complexes has suffered a revolution in the last years. Single Particle Analysis SPA and electron tomography are the two main branches responsible of this revolution. In both cases, the result is a 3D structure of the specimen. However, it is critical to determine the degree of reliability of the reconstructed structure, problem which is solved with resolution measurements. Resolution can be determined as a global parameter of the structure, or as a local property that can spatially vary. The main objective of the present thesis is the study of the local resolution and its applications for the validation of the reconstructed 3D structure and for local sharpening. For that, a mathematical method named MonoRes was developed, which measures the local resolution of reconstructed density maps and its algorithm represents the cornerstone of this thesis. Hence, MonoDir was designed for identifying reconstructions problems like the existence of preferred directions, or alignment errors. Note that the current state of the art does not provide an immediate and simultaneous response to both questions, in particular to the second one, which can be due to a systematic error of reconstruction. The proposed method does it by analyzing local-resolution anisotropy using the information of only the reconstructed map. Moreover, the measurement and analysis of the local resolution for map sharpening can be also used to enhance the visualization of the protein structure keeping its structure factor. For that, method LocalDeBlur was developed, which modifies the local amplitudes of the different frequencies according to the local resolution values. Apart from that, other methods were developed using slight variations of MonoRes algorithmic core. One of these methods is MonoTomo, which has been developed as the first method of local resolution in Electron Tomography. The estimation of the local resolution in electron tomograms involves work with spatially dependent noise and very large maps, so the core of MonoRes was extended in that direction, resulting in method MonoTomo, which solves those drawbacks. These other MonoRes core-derived methods extend the concept of resolution in a local manner. However, resolution is not only a local parameter, but it must be considered a tensor and, therefore, it depends on the position (voxel) and the direction. Thus, the concept of local resolution is then enriched with the notion of directionality.La crio-microscopía electrónica (cryoem) orientada a elucidar la estructura de complejos macromoleculares ha sufrido una considerable revolución en los últimos años. Los principales responsables de esta revolución son las técnicas de análisis de partículas individuales (Single Particle Analysis – SPA) y tomografía electrónica. En ambas, el resultado es obtención de estructura tridimensional del especimen. Sin embargo, es crítico determinar el grado de fiabilidad de la estructura reconstruida, este problema se soluciona gracias a las medidas de resolución. La resolución puede ser determinada como un parámetro global o como una propiedad local que varía espacialmente. El principal objetivo de esta tesis es el estudio de la resolución local y sus aplicaciones para la validación de las estructuras reconstruidas, así como su uso para “sharpening” local. Para ello hemos desarrollado un método, llamado MonoRes, que permite medir la resolución local de los mapas de densidad reconstruidos, este algoritmo representa el elemento central de la tesis. Así, otro algoritmo, MonoDir, fue diseñado para identificar problemas de reconstrución como pueden ser la existencia de direcciones preferentes de la muestra o errores de alineamiento. Nótese que el actual estado del arte no responder de manera inmediata y simultánea a ambas problemas de reconstrucción, en particular al segundo que puede ser debido a errores sistematicos. El método propuesto logra este acometido analizando la anisotropía de resolución local usando únicamente el mapa reconstruido. La medida y análisis de la resolución local puede emplear para realizar un sharpening del mapa y mejorar la visualización de la proteina manteniendo su factor de estructura. Para ello, fue desarrollado otro método llamado LocalDeBlur, el cual modifica las amplitudes de las diferentes frecuencias que contribuyen al mapa, pero teniendo en cuenta la información de la resolución local. Así mismo, también se muestran otros métodos usando variaciones del algoritmo de MonoRes. Uno de ellos es MonoTomo, el cual representa el primer algoritmo de resolución local en tomografía electrónica. La estimación de la resolución local in tomografía electrónica involucra trabajar con ruido espacialmente dependiente y mapas de gran tamaño. Los métodos propuestos extienden el concepto de resolución no solo de global a local, sino que muestran que la resolución es un tensor mostrando dependencia de la posición así como de la dirección, enriqueciendo todavía más la medida

Spline projection-based volume-to-image registration

This thesis focuses on the rigid-body registration of a three-dimensional model of an object to a set of its two-dimensional projections. The main contribution is the development of two registration algorithms that use a continuous model of the volume based on splines, either in the space domain or in the frequency domain. This allows for a well-defined gradient of the dissimilarity measure, which is a necessary condition for efficient and accurate registration. The first part of the thesis contains a review of the literature on volume-to- image registration. Then, we discuss data interpolation in the space domain and in the frequency domain. The basic concepts of our registration strategy are given in the second part of the thesis. We present a novel one-step approach for fast ray casting to simulate space-based volume projections. We also discuss the use of the central-slice theorem to simulate frequency-based volume projections. Then, we consider the question of the registration robustness. To improve the robustness of the space-based approach, we apply a multiresolution optimization strategy where spline-based data pyramids are processed in coarse-to-fine fashion, which improves speed as well. To improve the robustness of the frequency-based registration, we apply a coarse-to-fine strategy that involves weights in the frequency domain. In the third part, we apply our space-based algorithm to computer-assisted orthopedic surgery while adapting it to the perspective projection model. We show that the registration accuracy achieved using the orthopedic data is consistent with the current standards. Then, we apply our frequency-based registration to three-dimensional electron-microscopy application. We show that our algorithm can be used to obtain a refined solution with respect to currently available algorithms. The novelty of our approach is in dealing with a continuous space of geometric parameters, contrary to the standard methods which deal with quantized parameters. We conclude that our continuous parameter space leads to better registration accuracy. Last, we compare the performance of the frequency-based algorithm with that of the space-based algorithm in the context of electron microscopy. With these data, we observe that frequency-based registration algorithm outperforms the space-based one, which we attribute to the suitability of interpolation in the frequency domain when dealing with strictly space-limited data

Structure Characterization of the 70S-BipA Complex Using Novel Methods of Single-Particle Cryo-Electron Microscopy

Diseases caused by pathogenic bacteria continue to be major health concerns. For example, it is estimated that in the year 2000 typhoid fever caused over 21,000,000 illnesses and ~200,000 deaths (Crump et al., 2004). The disease is caused by S. typhi, a closely-related serotype of S. typhiumurium, the salmonella strain in which BipA was first identified. The CDC estimated that in 2013, multidrug resistant bacteria caused over 2 million infections in the United States, ending in more than 23,000 deaths (CDC, 2013). This number is set to rise as more bacteria become resilient to the collection of conventional antibiotics. The increasing number of multidrug resistant bacterial strains necessitates the development of new antimicrobial drugs. BipA is an attractive target for drug research. As mentioned in Section 2.5.2, BipA is ubiquitous in eubacteria and lower eukaryotes such as protozoa, but is absent from higher-order eukaryotes such as humans. Because the protein is essential for bacterial survival, BipA presents a major vulnerability of pathogenic bacteria. A drug targeting the protein itself or its interactions to the ribosome will disable only the bacteria, but have no effect on the eukaryotic host. A comprehensive model of BipA bound to the 70S ribosome will provide unparalleled insight into BipA's binding site and its mechanism. Toward this goal, cryo-EM techniques were employed to visualize the binding site of BipA on the 70S ribosome, characterize its interactions with the ribosome, and elucidate its mechanism on the ribosome. An X-ray structure of isolated BipA-GMPPNP was elucidated, by collaborators, and used for further molecular modeling of the protein to reveal possible atomic interactions between BipA and 70S ribosome. Additional biochemical studies were performed to fully characterize the specific ribosomal complex that optimizes binding of the factor. Together, the cryo-EM reconstruction, the BipA X-ray structure, the subsequent molecular modeling, and the additional biochemical studies provide a comprehensive model for BipA binding. Over the last years, the introduction of new automated algorithms for particle selection (AutoPicker) and classification (RELION) for the cryo-EM technique has revolutionized the workflow of the entire imaging and reconstruction process. The BipA dataset was primed to be used as a test bed for these algorithms and classification technique, respectively. Using old and new techniques to process the dataset allows a discussion of how the single particle reconstruction process can be vastly improved, with greater automation and efficiency

Étude structurale de deux complexes macromoléculaires biologiques : FANCD2/FANCI et la Phosphorylase Kinase par cryo microscopie électronique

During my thesis work, I have investigated the structure of two protein complexes, the FANCD2/FANCI complex and the Phosphorylase Kinase complex (PhK). Both complexes were studied using cryo electron microscopy combined with image analysis. The Fanconi Anemia (FA) pathway has been implied to play a significant role in DNA interstrand crosslink repair and may be the coordinator between different DNA damage repair pathways. Within the FA pathway, the FANCD2 and FANCI proteins are key players. In my thesis work, I have calculated the structure of the human FANCD2/FANCI complex. It possesses an inner cavity, large enough to accommodate a double stranded DNA helix. We also discovered a protruding tower domain, which our collaborator (M. Cohn, Oxford) has shown to be critical for the recruitment of the complex to DNA. PhK is one of the most complex kinases. It is composed of four subunits (αβγδ)4. PhK regulates glycogenolysis, it integrates various signals to catalyze the conversion of glycogen phosphorylase (GP) b to GP a (active), and the subsequent breakdown of glycogen. PhK is a potential target for glycemic control in diseases such as diabetes. Using state of the art electron microscope with a direct electron detection camera, after multiple image processing steps and correction of beams induced motion of films, I obtained a structure of the complexe at 7Å (FSC gold standard).Au cours de mon travail de thèse, j’ai étudié la structure des deux complexes de protéines, le complexe FANCD2/FANCI et la Phosphorylase kinase (PhK). Les deux complexes ont été étudiés en utilisant la cryo-microscopie électronique combinée à l’analyse d'image. La voie anémie de Fanconi (FA) a été reconnue comme jouant un rôle important dans la réparation de liaisons inter-brin de l'ADN. Dans cette voie, les protéines FANCD2 et FANCI sont des acteurs clés. Dans mon travail de thèse, j’ai calculé la structurale du complexe FANCD2/FANCI humaine. La structure montre une cavité intérieure, assez grande pour accueillir une hélice d'ADN double brin. Nous avons aussi mis en évidence un domaine en forme de tour. Notre collaborateur (M. Cohn, Oxford) a montré que celui-ci est essentiel pour le recrutement du complexe sur l'ADN. La PhK est l'une des kinases les plus complexes. Elle est composée de quatre sous-unités (αβγδ)4. PhK régule le métabolisme du glycogène, intègre divers signaux pour catalyser la conversion du glycogène phosphorylase b (GP) vers la GP a (actif), et la dégradation ultérieure de glycogène. En utilisant un microscope performant et une caméra de détection d'électrons directe puis après plusieurs étapes de traitement d’image, de correction de mouvement de films induits par les faisceaux d'électrons, j’ai obtenu une structure du complexe en 7Å (FSC gold standard)

New computational methods toward atomic resolution in single particle cryo-electron microscopy

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Escuela Politécnica Superior, Departamento de Ingeniería Informática. Fecha de lectura: 22-06-2016Structural information of macromolecular complexes provides key insights into the way they carry out their biological functions. In turn, Electron microscopy (EM) is an essential tool to study the structure and function of biological macromolecules at a medium-high resolution. In this context, Single-Particle Analysis (SPA), as an EM modality, is able to yield Three-Dimensional (3-D) structural information for large biological complexes at near atomic resolution by combining many thousands of projection images. However, these views su er from low Signal-to-Noise Ratios (SNRs), since an extremely low total electron dose is used during exposure to reduce radiation damage and preserve the functional structure of macromolecules. In recent years, the emergence of Direct Detection Devices (DDDs) has opened up the possibility of obtaining images with higher SNRs. These detectors provide a set of frames instead of just one micrograph, which makes it possible to study the behavior of frozen hydrated specimens as a function of electron dose and rate. In this way, it has become apparent that biological specimens embedded in a solid matrix of amorphous ice move during imaging, resulting in Beam-Induced Motion (BIM). Therefore, alignment of frames should be added to the classical standard data processing work ow of single-particle reconstruction, which includes: particle selection, particle alignment, particle classi cation, 3-D reconstruction, and model re nement. In this thesis, we propose new algorithms and improvements for three important steps of this work ow: movie alignment, particles selection, and 3-D reconstruction. For movie alignment, a methodology based on a robust to noise optical ow approach is proposed that can e ciently correct for local movements and provide quantitative analysis of the BIM pattern. We then introduce a method for automatic particle selection in micrographs that uses some new image features to train two classi ers to learn from the user the kind of particles he is interested in. Finally, for 3-D reconstruction, we introduce a gridding-based direct Fourier method that uses a weighting technique to compute a uniform sampled Fourier transform. The algorithms are fully implemented in the open-source Xmipp package (http://xmipp.cnb.csic.es