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

Predicting multibody assembly of proteins

textThis thesis addresses the multi-body assembly (MBA) problem in the context of protein assemblies. [...] In this thesis, we chose the protein assembly domain because accurate and reliable computational modeling, simulation and prediction of such assemblies would clearly accelerate discoveries in understanding of the complexities of metabolic pathways, identifying the molecular basis for normal health and diseases, and in the designing of new drugs and other therapeutics. [...] [We developed] F²Dock (Fast Fourier Docking) which includes a multi-term function which includes both a statistical thermodynamic approximation of molecular free energy as well as several of knowledge-based terms. Parameters of the scoring model were learned based on a large set of positive/negative examples, and when tested on 176 protein complexes of various types, showed excellent accuracy in ranking correct configurations higher (F² Dock ranks the correcti solution as the top ranked one in 22/176 cases, which is better than other unsupervised prediction software on the same benchmark). Most of the protein-protein interaction scoring terms can be expressed as integrals over the occupied volume, boundary, or a set of discrete points (atom locations), of distance dependent decaying kernels. We developed a dynamic adaptive grid (DAG) data structure which computes smooth surface and volumetric representations of a protein complex in O(m log m) time, where m is the number of atoms assuming that the smallest feature size h is [theta](r[subscript max]) where r[subscript max] is the radius of the largest atom; updates in O(log m) time; and uses O(m)memory. We also developed the dynamic packing grids (DPG) data structure which supports quasi-constant time updates (O(log w)) and spherical neighborhood queries (O(log log w)), where w is the word-size in the RAM. DPG and DAG together results in O(k) time approximation of scoring terms where k << m is the size of the contact region between proteins. [...] [W]e consider the symmetric spherical shell assembly case, where multiple copies of identical proteins tile the surface of a sphere. Though this is a restricted subclass of MBA, it is an important one since it would accelerate development of drugs and antibodies to prevent viruses from forming capsids, which have such spherical symmetry in nature. We proved that it is possible to characterize the space of possible symmetric spherical layouts using a small number of representative local arrangements (called tiles), and their global configurations (tiling). We further show that the tilings, and the mapping of proteins to tilings on arbitrary sized shells is parameterized by 3 discrete parameters and 6 continuous degrees of freedom; and the 3 discrete DOF can be restricted to a constant number of cases if the size of the shell is known (in terms of the number of protein n). We also consider the case where a coarse model of the whole complex of proteins are available. We show that even when such coarse models do not show atomic positions, they can be sufficient to identify a general location for each protein and its neighbors, and thereby restricts the configurational space. We developed an iterative refinement search protocol that leverages such multi-resolution structural data to predict accurate high resolution model of protein complexes, and successfully applied the protocol to model gp120, a protein on the spike of HIV and currently the most feasible target for anti-HIV drug design.Computer Science

Structural studies of influenza A virus by cryo-electron tomography

Influenza A virus (IAV) is a pleomorphic, enveloped virus known for its yearly epidemics and occasional, but fatal pandemics. The outer surface glycoprotein hemagglutinin (HA) together with the matrix protein 1 (M1) are the most abundant protein components of assembled virions. HA, located at the outside of virions, is involved in cell receptor recognition, membrane fusion and is the most relevant protein for antibody binding. Therefore the structure of isolated HA has been extensively characterised by X-ray crystallography. However, it remains unclear to which extent the structure of isolated HA corresponds to the in situ HA structure on the surface of IAV. M1 determines the morphology of the virus by forming a matrix layer underneath the viral membrane. A high resolution structure of full length M1 is missing and the lack of information about the in situ arrangement of the M1 matrix layer currently limits our understanding of how M1 functions. Here, I set out to determine the structures of HA and M1 directly from IAV particles using high resolution cryo-electron tomography (cryoET) and subtomogram averaging. I found that virus purification can affect the integrity of the virus HA glycoprotein layer and the morphology of virus particles. I therefore adapted a workflow which allows studying the structure of viral proteins directly from viruses in the vicinity of virus-producing cells. Biosafety regulations required inactivation of IAV samples by chemical fixation prior to cryoEM imaging. To assess effects of fixation, I complemented structural studies of HA from pathogenic, fixed IAV particles with studies of HA from non-infectious, unfixed virus-like particles (VLPs). These studies revealed that fixation captures HA in an open conformation while HA structures determined from unfixed samples perfectly match the closed conformation observed in the trimeric crystal structure. In concordance with recent work by others, this observation suggests that fixation captures HA in a an open, otherwise transient conformation, which is part of a constant opening and closing motion known as breathing motion. To characterise the in situ structure and arrangement of M1, I established a subtomogram averaging workflow to cope with the challenges presented by the small size of M1. I successfully obtained two independent structures of M1 directly from viruses and VLPs. Comparisons of my structures to existing high resolution models of the N-terminal domain (NTD) of M1 revealed that M1 monomers arrange as parallel strands, with a helical propensity and directly underneath the membrane. For the first time, my data allow to describe the M1-membrane interface as well as relevant M1-M1 interfaces within the matrix layer. Finally, I have gained first structural insights into the M1 C-terminal domain (CTD). I further combined the obtained structural information for M1 with a theoretical model of the mechanics of M1 polymerization and membrane deformation during virus assembly. The obtained results suggest that linear polymerization of M1 into multiple parallel strands efficiently provides energy to drive assembly of new virus particles. The results presented in this thesis improve our understanding of the arrangement and structure of the two influenza proteins HA and M1 in situ which has implications for current models of HA-mediated membrane fusion, virus architecture and virus assembly

Discrete element modelling of permanent pavement deformation in granular materials

The permanent deformation of a pavement due to vehicle load is one of the important factors affecting the design life as well as the maintenance cost of a pavement. For the purpose of obtaining a cost-effective design, it is advisable to predict the traffic-loadinduced permanent pavement deformation. The permanent deformation in pavements (i.e. rutting) can be classified into three categories, including the wearing of the asphalt layers, compaction, and shear deformations. In the present study, discrete element analyses have been performed to predict the permanent deformation of a pavement when subjected to moving wheel loads. Note that the wearing of the asphalt layers has been disregarded. DEM biaxial test simulations have been carried out in terms of both unbonded and bonded granular materials. The typical stress-strain response, as well as the volumetric strain development, have been reproduced, in qualitative agreement with the experimental results. The factors affecting the mechanical behaviour of granular materials have been investigated, e.g. particle stiffness, sample compaction and parallel bond strength. In addition, the elastic properties, initial yield stress, strength parameters and so on have been analysed. These compression tests provided guidance for the selection of the particle parameters for the subsequent pavement simulation. The permanent deformation in unbonded pavements was represented under moving wheel loads, and proved to be qualitatively consistent with the laboratory tests. The initial self-weight stress had a significant effect on rutting. When the initial gravity stress was relatively high, both shakedown and surface ratchetting phenomena were observed for different loading levels. However, the accumulation of permanent deformation was continual for pavements with low gravity stress, even if the wheel pressure was small. Other factors affecting the rutting have been taken into consideration, e.g. specimen preparation, interparticle friction, etc. In the case of the single-layered pavement, permanent deformation ceased after the first wheel pass. Plastic deformation increased with the decrease in the self-weight stress. For the double layered pavement, the permanent deformation continually increased with wheel passes, probably owing to compaction of the bottom unbound layer. The pavement shakedown phenomenon was not observed prior to wheel pass 300. The permanent deformation increased augmentation of wheel pressure as well as decrease in the sample density and upper layer thickness. The residual stresses in both vertical and horizontal directions can be obtained using the measurement circle. For all the pavements in the current simulations, the vertical residual stress is nearly always zero, consistent with the equilibrium condition. In the case of the unbonded pavement, the large horizontal residual stress depends on the high initial gravity stress, instead of high wheel pressure or wheel pass number. For the single-layered pavement, the peak of the horizontal residual stress was observed near the pavement surface. The residual stress rises with the augmentation of the wheel pass number and the wheel pressure. In the double-layered pavement, the residual stresses are discontinuous at the interface between different pavement layers. The peak appears near the pavement surface and increases with the reduction in the upper layer thickness as well as the rise in wheel passes and wheel pressure. Nevertheless, residual stress is not apparent in the granular base. The probability density distribution was investigated in terms of the contact and bond forces. For the normal contact force, a peak generally appeared at small contact forces, followed by a drastic decrease and, after that, the probability density progressively approached zero. For the tangential contact force as well as the bond forces, in general, a peak of the probability distribution was observed at small contact forces, and then a sharp drop followed from the two flanks of the peak point. Finally, there was a gradual decrease until the probability density decayed to zero. The factors, e.g. pavement layer, wheel pass number and wheel pressure, mainly affect the probability distribution of the small contact or bond forces. For both single- and double-layered pavements, the absolute extrema of the bond forces in the top layer increased with the augmentation of the wheel pass number and the wheel pressure. For the unbonded pavement, the sliding contact ratio was studied and it was significantly affected by the pavement layer, initial gravity stress and sample compaction. The distribution of the pavement particle displacements were demonstrated. In the unbonded pavement, factors, such as wheel pressure and initial gravity, not only affect the distribution of the relatively large particle displacements but also increase the magnitude of the particle displacements. The directions of the large displacement vectors are diverse as the large gravity acceleration is assigned to the particles but are almost downward when the self-weight stress is small. In the single- or double layered pavement, factors, such as wheel pass number and wheel pressure, merely increase the values of the particle displacements. The distribution of the displacements is hardly affected. For the single-layered pavement, the large displacements were observed near the pavement surface and their directions are almost contrary to the movement direction of the wheel. In the double-layered pavement, relatively large particle displacements are widely distributed in the pavement. Their directions are in an almost vertical direction

Discrete element modelling of permanent pavement deformation in granular materials

The permanent deformation of a pavement due to vehicle load is one of the important factors affecting the design life as well as the maintenance cost of a pavement. For the purpose of obtaining a cost-effective design, it is advisable to predict the traffic-loadinduced permanent pavement deformation. The permanent deformation in pavements (i.e. rutting) can be classified into three categories, including the wearing of the asphalt layers, compaction, and shear deformations. In the present study, discrete element analyses have been performed to predict the permanent deformation of a pavement when subjected to moving wheel loads. Note that the wearing of the asphalt layers has been disregarded. DEM biaxial test simulations have been carried out in terms of both unbonded and bonded granular materials. The typical stress-strain response, as well as the volumetric strain development, have been reproduced, in qualitative agreement with the experimental results. The factors affecting the mechanical behaviour of granular materials have been investigated, e.g. particle stiffness, sample compaction and parallel bond strength. In addition, the elastic properties, initial yield stress, strength parameters and so on have been analysed. These compression tests provided guidance for the selection of the particle parameters for the subsequent pavement simulation. The permanent deformation in unbonded pavements was represented under moving wheel loads, and proved to be qualitatively consistent with the laboratory tests. The initial self-weight stress had a significant effect on rutting. When the initial gravity stress was relatively high, both shakedown and surface ratchetting phenomena were observed for different loading levels. However, the accumulation of permanent deformation was continual for pavements with low gravity stress, even if the wheel pressure was small. Other factors affecting the rutting have been taken into consideration, e.g. specimen preparation, interparticle friction, etc. In the case of the single-layered pavement, permanent deformation ceased after the first wheel pass. Plastic deformation increased with the decrease in the self-weight stress. For the double layered pavement, the permanent deformation continually increased with wheel passes, probably owing to compaction of the bottom unbound layer. The pavement shakedown phenomenon was not observed prior to wheel pass 300. The permanent deformation increased augmentation of wheel pressure as well as decrease in the sample density and upper layer thickness. The residual stresses in both vertical and horizontal directions can be obtained using the measurement circle. For all the pavements in the current simulations, the vertical residual stress is nearly always zero, consistent with the equilibrium condition. In the case of the unbonded pavement, the large horizontal residual stress depends on the high initial gravity stress, instead of high wheel pressure or wheel pass number. For the single-layered pavement, the peak of the horizontal residual stress was observed near the pavement surface. The residual stress rises with the augmentation of the wheel pass number and the wheel pressure. In the double-layered pavement, the residual stresses are discontinuous at the interface between different pavement layers. The peak appears near the pavement surface and increases with the reduction in the upper layer thickness as well as the rise in wheel passes and wheel pressure. Nevertheless, residual stress is not apparent in the granular base. The probability density distribution was investigated in terms of the contact and bond forces. For the normal contact force, a peak generally appeared at small contact forces, followed by a drastic decrease and, after that, the probability density progressively approached zero. For the tangential contact force as well as the bond forces, in general, a peak of the probability distribution was observed at small contact forces, and then a sharp drop followed from the two flanks of the peak point. Finally, there was a gradual decrease until the probability density decayed to zero. The factors, e.g. pavement layer, wheel pass number and wheel pressure, mainly affect the probability distribution of the small contact or bond forces. For both single- and double-layered pavements, the absolute extrema of the bond forces in the top layer increased with the augmentation of the wheel pass number and the wheel pressure. For the unbonded pavement, the sliding contact ratio was studied and it was significantly affected by the pavement layer, initial gravity stress and sample compaction. The distribution of the pavement particle displacements were demonstrated. In the unbonded pavement, factors, such as wheel pressure and initial gravity, not only affect the distribution of the relatively large particle displacements but also increase the magnitude of the particle displacements. The directions of the large displacement vectors are diverse as the large gravity acceleration is assigned to the particles but are almost downward when the self-weight stress is small. In the single- or double layered pavement, factors, such as wheel pass number and wheel pressure, merely increase the values of the particle displacements. The distribution of the displacements is hardly affected. For the single-layered pavement, the large displacements were observed near the pavement surface and their directions are almost contrary to the movement direction of the wheel. In the double-layered pavement, relatively large particle displacements are widely distributed in the pavement. Their directions are in an almost vertical direction

High resolution three-dimensional reconstruction for small animal PET cameras

Tesis doctoral inédita. Universidad Autónoma de Madrid, Escuela Politécnica Superior, junio de 201

Microscopy Conference 2017 (MC 2017) - Proceedings

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2017", die vom 21. bis 25.08.2017, in Lausanne stattfand

Microscopy Conference 2017 (MC 2017) - Proceedings

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2017", die vom 21. bis 25.08.2017, in Lausanne stattfand