Unsupervised cryo-EM data clustering through adaptively constrained K-means algorithm

In single-particle cryo-electron microscopy (cryo-EM), K-means clustering algorithm is widely used in unsupervised 2D classification of projection images of biological macromolecules. 3D ab initio reconstruction requires accurate unsupervised classification in order to separate molecular projections of distinct orientations. Due to background noise in single-particle images and uncertainty of molecular orientations, traditional K-means clustering algorithm may classify images into wrong classes and produce classes with a large variation in membership. Overcoming these limitations requires further development on clustering algorithms for cryo-EM data analysis. We propose a novel unsupervised data clustering method building upon the traditional K-means algorithm. By introducing an adaptive constraint term in the objective function, our algorithm not only avoids a large variation in class sizes but also produces more accurate data clustering. Applications of this approach to both simulated and experimental cryo-EM data demonstrate that our algorithm is a significantly improved alterative to the traditional K-means algorithm in single-particle cryo-EM analysis.Comment: 35 pages, 14 figure

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 achievedWe acknowledge support from “la Caixa” Foundation (Fellowship LCF/BQ/DI18/11660021. This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 713673. We also thank the financial support from the Spanish Ministry of Economy and Competitiveness through Grants BIO2016-76400-R(AEI/FEDER, UE) and SEV 2017-0712, the “Comunidad Autónoma de Madrid” through Grant: S2017/BMD-3817, Instituto de Salud Carlos III, PT17/ 0009/0010 (ISCIII-SGEFI/ERDF), European Union (EU) and Horizon 2020 through grants: CORBEL (INFRADEV-1-2014-1, Proposal: 654248), INSTRUCT-ULTRA (INFRADEV-03-2016-2017, Proposal: 731005), EOSC Life (INFRAEOSC-04-2018, Proposal: 824087), High- ResCells (ERC-2018-SyG, Proposal: 810057), IMpaCT (WIDESPREAD-03-2018 – Proposal: 857203), EOSC-Synergy (EINFRA-EOSC-5, Proposal: 857647), and iNEXT-Discovery (Proposal: 871037). The authors acknowledge the support and the use of resources of Instruct, a Landmark ESFRI projec

Particle alignment reliability in single particle electron cryomicroscopy: A general approach

Electron Microscopy is reaching new capabilities thanks to the combined effect of new technologies and new image processing methods. However, the reconstruction process is still complex, requiring many steps and elaborated optimization procedures. Therefore, the possibility to reach a wrong structure exists, justifying the need of robust statistical tests. In this work, we present a conceptually simple alignment test, which does not require tilt-pair images, to evaluate the alignment consistency between a set of projection images with respect to a given 3D density map. We test the approach on a number of problems in 3DEM, especially the ranking and evaluation of initial 3D volumes and high resolution 3D maps, where we show its usefulness in providing an objective evaluation for maps that have recently been subject to a strong controversy in the field. Additionally, this alignment statistical test can be linked to the early stages of structure solving of new complexes, streamlining the whole process.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), and from "Comfuturo" grant funded by the Fundacion General CSIC. C.O.S. Sorzano is recipient of a Ramon y Cajal fellowship. We acknowledge discussions with Sriram Subramanian on validation approaches related to the HIV trimer structure

Fast and robust single particle reconstruction in 3D fluorescence microscopy

Single particle reconstruction has recently emerged in 3D fluorescence microscopy as a powerful technique to improve the axial resolution and the degree of fluorescent labeling. It is based on the reconstruction of an average volume of a biological particle from the acquisition multiple views with unknown poses. Current methods are limited either by template bias, restriction to 2D data, high computational cost or a lack of robustness to low fluorescent labeling. In this work, we propose a single particle reconstruction method dedicated to convolutional models in 3D fluorescence microscopy that overcome these issues. We address the joint reconstruction and estimation of the poses of the particles, which translates into a challenging non-convex optimization problem. Our approach is based on a multilevel reformulation of this problem, and the development of efficient optimization techniques at each level. We demonstrate on synthetic data that our method outperforms the standard approaches in terms of resolution and reconstruction error, while achieving a low computational cost. We also perform successful reconstruction on real datasets of centrioles to show the potential of our method in concrete applications

Pea PSII-LHCII supercomplexes form pairs by making connections across the stromal gap

In higher plant thylakoids, the heterogeneous distribution of photosynthetic protein complexes is a determinant for the formation of grana, stacks of membrane discs that are densely populated with Photosystem II (PSII) and its light harvesting complex (LHCII). PSII associates with LHCII to form the PSII-LHCII supercomplex, a crucial component for solar energy conversion. Here, we report a biochemical, structural and functional characterization of pairs of PSII-LHCII supercomplexes, which were isolated under physiologically-relevant cation concentrations. Using single-particle cryo-electron microscopy, we determined the three-dimensional structure of paired C2S2M PSII-LHCII supercomplexes at 14 angstrom resolution. The two supercomplexes interact on their stromal sides through a specific overlap between apposing LHCII trimers and via physical connections that span the stromal gap, one of which is likely formed by interactions between the N-terminal loops of two Lhcb4 monomeric LHCII subunits. Fast chlorophyll fluorescence induction analysis showed that paired PSII-LHCII supercomplexes are energetically coupled. Molecular dynamics simulations revealed that additional flexible physical connections may form between the apposing LHCII trimers of paired PSII-LHCII supercomplexes in appressed thylakoid membranes. Our findings provide new insights into how interactions between pairs of PSII-LHCII supercomplexes can link adjacent thylakoids to mediate the stacking of grana membranes

Structural analysis of macromolecular nanomachines by 3d electron microscopy: managing flexibility and heterogenety in some define cases.

The term macromolecular nanomachines refers to those complexes that correspond to the functional machinery in the context of the living cell,, from DNA repair or replication, to protein degradation or cell división, to name just a few functions. Normally, they are formed by different types of proteins and may also include nucleic acids. The estructure of macromolecular nanomachines and their structural changes is a very important process with a vital role in functional implications. Their study is many times accomplished by three-dimensional elecron microscopy, since they are too large for Nuclear Magnetic Resonance and too flexible for X-ray Diffraction. In these termes, Electron microscopy has provided important structdural information about protein complexes essential for describing their functional organization. Understanding how function occurs can only be inferred after integrating many different types of dataEl trabajo recogido en esta memoria ha sido realizado en el Centro Nacional de Biotecnología (CNB-CSIC) bajo la dirección conjunta de los Drs. José María Carazo García y Carlos Oscar Sorzano Sánchez. Su financiación corrió a cargo de una beca predoctoral de Formación de Personal Investigador (FPI) y de los proyectos Instruct, CAM(S2010/BMD-2305), AIC-A-2011-0638 y AICA-2011-0638

Aceleración de algoritmos de procesamiento de imágenes para el análisis de partículas individuales con microscopia electrónica

Tesis Doctoral inédita cotutelada por la Masaryk University (República Checa) y la Universidad Autónoma de Madrid, Escuela Politécnica Superior, Departamento de Ingeniería Informática. Fecha de Lectura: 24-10-2022Cryogenic Electron Microscopy (Cryo-EM) is a vital field in current structural biology. Unlike X-ray crystallography and Nuclear Magnetic Resonance, it can be used to analyze membrane proteins and other samples with overlapping spectral peaks. However, one of the significant limitations of Cryo-EM is the computational complexity. Modern electron microscopes can produce terabytes of data per single session, from which hundreds of thousands of particles must be extracted and processed to obtain a near-atomic resolution of the original sample. Many existing software solutions use high-Performance Computing (HPC) techniques to bring these computations to the realm of practical usability. The common approach to acceleration is parallelization of the processing, but in praxis, we face many complications, such as problem decomposition, data distribution, load scheduling, balancing, and synchronization. Utilization of various accelerators further complicates the situation, as heterogeneous hardware brings additional caveats, for example, limited portability, under-utilization due to synchronization, and sub-optimal code performance due to missing specialization. This dissertation, structured as a compendium of articles, aims to improve the algorithms used in Cryo-EM, esp. the SPA (Single Particle Analysis). We focus on the single-node performance optimizations, using the techniques either available or developed in the HPC field, such as heterogeneous computing or autotuning, which potentially needs the formulation of novel algorithms. The secondary goal of the dissertation is to identify the limitations of state-of-the-art HPC techniques. Since the Cryo-EM pipeline consists of multiple distinct steps targetting different types of data, there is no single bottleneck to be solved. As such, the presented articles show a holistic approach to performance optimization. First, we give details on the GPU acceleration of the specific programs. The achieved speedup is due to the higher performance of the GPU, adjustments of the original algorithm to it, and application of the novel algorithms. More specifically, we provide implementation details of programs for movie alignment, 2D classification, and 3D reconstruction that have been sped up by order of magnitude compared to their original multi-CPU implementation or sufficiently the be used on-the-fly. In addition to these three programs, multiple other programs from an actively used, open-source software package XMIPP have been accelerated and improved. Second, we discuss our contribution to HPC in the form of autotuning. Autotuning is the ability of software to adapt to a changing environment, i.e., input or executing hardware. Towards that goal, we present cuFFTAdvisor, a tool that proposes and, through autotuning, finds the best configuration of the cuFFT library for given constraints of input size and plan settings. We also introduce a benchmark set of ten autotunable kernels for important computational problems implemented in OpenCL or CUDA, together with the introduction of complex dynamic autotuning to the KTT tool. Third, we propose an image processing framework Umpalumpa, which combines a task-based runtime system, data-centric architecture, and dynamic autotuning. The proposed framework allows for writing complex workflows which automatically use available HW resources and adjust to different HW and data but at the same time are easy to maintainThe project that gave rise to these results received the support of a fellowship from the “la Caixa” Foundation (ID 100010434). The fellowship code is LCF/BQ/DI18/11660021. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 71367

Structure and structural dynamics of Photosystem II supercomplex in higher plants

Photosynthesis is indisputably the primary biological process to introduce chemical energy and biomass into ecosystems by oxidizing water and reducing carbon dioxide into organic compounds. Photosystem II (PSII) is a unique protein complex, present in thylakoid membranes of all oxygenic photosynthetic organisms, able to catalyze the water-splitting reaction using sunlight as driving force, thus being responsible for the generation of all the molecular oxygen accumulated in the atmosphere for over three billion years. Although its catalytic core has been extremely conserved throughout evolution, from cyanobacteria to higher plants, the necessity of different photosynthetic organisms to cope with ever-changing environmental light conditions led to the emergence of a great variability among its peripheral antenna systems, differentiating in extrinsic phycobilisomes in cyanobacteria and intrinsic light harvesting complexes (LHCII) in green algae and higher plants. LHCII are integral membrane proteins that occur as heterotrimers of Lhcb1-2-3 subunits and monomeric Lhcb4-5-6 polypeptides and associate peripherally with the PSII core in variable numbers, thus forming large supramolecular assemblies called PSII‐LHCII supercomplexes. The minimal functional unit, found in all light conditions, consists of a dimeric PSII core (C2) with two strongly bound LHCII trimers (S2), made of Lhcb1 and Lhcb2, connected by two monomeric Lhcb4 and Lhcb5 subunits, and is called C2S2. In limiting light conditions, the C2S2 can further associate with one or two moderately bound LHCII trimers (M2) which consist of Lhcb1, Lhcb2 and Lhcb3 proteins connected by the monomeric Lhcb6, a peculiar subunit found only in higher plants, originating supercomplexes of type C2S2M1-2. A further supramolecular organization is due to the lateral association of PSII-LHCII supercomplexes within the thylakoid membrane plane, forming PSII-LHCII megacomplexes, or even higher ordered arrays. The LHCII fulfill a dual role by either quenching the excess light energy, often occurring in natural environments, or optimizing its harvesting in ecosystems where there is competition and mutual shading. The rearrangement of the PSII’s modular antenna system through its dynamic interaction with the PSII core, therefore, appears to be a key process in light harvesting regulation. Moreover, plant’s PSII and LHCII are spatially and functionally segregated into piled discs of thylakoid membranes (grana), where they occupy 80% of the surface. Their structural arrangement into PSII-LHCII supercomplexes interacting dynamically with each other appears to be critical in determining the overall membrane architecture and ultimately the efficiency of photosynthesis. Although the overall structure of the basic C2S2 supercomplex in plants has been recently resolved at nearly atomic resolution, there is still a lack of knowledge regarding its structural rearrangement in different light conditions as well as its specific interaction within the membrane plane and between adjacent membranes. During this thesis’ work we have been able to isolate pure PSII-LHCII super- and megacomplexes from pea plants grown in moderate light by mild solubilization of stacked thylakoid membranes. In order to assess their overall functional architecture, the full biochemical characterization of isolated PSII-LHCII supercomplexes, comprehensive of accurate proteomic analyses, was coupled with structural studies. Their structural characterization, performed by transmission electron microscopy (TEM) in cryogenic conditions (cryo-EM) and subsequent single particle analysis, led to a novel 3D structure at about 14 Å resolution of the supercomplexes of type C2S2M. The obtained electron density map revealed that under normal light conditions most of the supercomplexes within the grana are of type C2S2M and occur as paired supercomplexes, whose interactions are mediated by physical connections across the stromal gap of adjacent membranes. The specific overlapping of LHCII trimers facing each other in paired supercomplexes, as already observed in other studies, suggests that this conformation might be representative of their native state within the membranes. The physical connections observed across the stromal gap might be attributable to the mutual interaction between the long N-terminal loops of the monomeric Lhcb4 subunits. These subunits occupy a pivotal position in the 3D map of the paired supercomplexes and are clearly bridged across the stromal gap by electron densities attributable to these loops. In addition, despite the its structural flexibility, the remarkable sequence conservation of this region, even in distant phylogenetic photosynthetic organisms, may suggest its major involvement in structural dynamics. The specific interaction observed in paired supercomplexes seems to be mediated by cations present within the chloroplast in relatively low concentrations as their depletion from buffers used for isolation leads to the dissociation of the paired supercomplexes into single ones. Moreover, this evidence was also strongly supported by the decrease in the PSII excitonic connectivity measured in-vivo. The paired behavior has also been observed in higher oligomerization forms of isolated PSII-LHCII supercomplexes in which two paired supercomplexes laterally interact with each other in the membrane plane, thus forming paired megacomplexes. This novel structure has been obtained by EM and 2D reconstruction of negatively stained particles and, despite its low resolution, reveals how PSII-LHCII supercomplexes may laterally and stromally interact with each other in different ways. The observation of the potential overlapping of LHCII trimers in megacomplexes facing each other, as well as the occurrence of different geometries of interaction between supercomplexes within the membrane plane and between megacomplexes in adjacent membranes, provide intriguing insights on how PSII and LHCII might interact in a very stable manner within the thylakoid membrane and between different discs in the grana. In order to study the PSII-LHCII supercomplex remodeling in the context of ever-changing light environmental conditions, PSII-LHCII supercomplexes have been isolated from pea plants grown at different light intensities: low (LL), moderate (CL) and high light (HL). The accurate profiling and quantitation of the LHCII subunits in the isolated supercomplexes and in the native thylakoids, achieved by using a mass-spectrometry based proteomic approach, was coupled with the evaluation in-vivo of their functional antenna size (ASII). At increasing light intensities, the structural remodeling of the modular PSII’s antenna system led to the reduction of the amount of LHCII M-trimers in the isolated complexes, attested by the decreased level of Lhcb3 and Lhcb6. This specific remodeling does not occur at the same rate in the entire thylakoid membrane. The whole LHCII pool is downregulated only in plants grown in HL, suggesting the occurrence of different acclimation strategies. The remarkable decrease of the ASII observed in HL acclimated plants, when compared to LL plants, can be attributed to the significant increase of the Lhcb4 specific isoform Lhcb4.3, occurring both in isolated supercomplexes and in thylakoid membranes. Unlike isoforms Lhcb4.1-2, the Lhcb4.3 isoform, whose transcription is enhanced upon HL exposure, interestingly has a truncated C-terminus that is located at the binding interface with Lhcb6 within the supercomplex structure. The incorporation of Lhcb4.3 in the PSII-LHCII supercomplex might play a major role in decreasing its functional antenna size by reducing its affinity to bind additional M-trimers, thus regulating its light harvesting efficiency even at moderate light intensities. Conversely, the exposure to HL induces the decrease of the PSII antenna cross-section in isolated supercomplexes and the partial depletion of the whole antenna system of PSII in the thylakoid membranes, thus constitutively preventing damages to the reaction center when light continuously exceeds its energy-processing capacity. These results aim at broadening the current knowledge on how the light harvesting antenna system associated with the PSII core is finely regulated upon plants’ long term acclimation to different light intensities. The flexibility of the PSII’s modular antenna system, accompanied by its finely tuned structural interaction with the core complex, pivotal for the 3D organization of plant thylakoid membranes, certainly played a key role in determining its remarkable evolutionary outcome. Taken together, these results may provide new research directions while certainly broadening the knowledge on how PSII-LHCII assemblies and their supramolecular interaction contribute to maintain the complex architecture of thylakoid membranes and the overall efficiency of photosynthesis in ever changing environmental conditions