RESUMEN
Most life scientists would agree that understanding how cellular processes work requires structural knowledge about the macromolecules involved. For example, deciphering the double-helical nature of DNA revealed essential aspects of how genetic information is stored, copied and repaired. Yet, being reductionist in nature, structural biology requires the purification of large amounts of macromolecules, often trimmed off larger functional units. The advent of cryogenic electron microscopy (cryo-EM) greatly facilitated the study of large, functional complexes and generally of samples that are hard to express, purify and/or crystallize. Nevertheless, cryo-EM still requires purification and thus visualization outside of the natural context in which macromolecules operate and coexist. Conversely, cell biologists have been imaging cells using a number of fast-evolving techniques that keep expanding their spatial and temporal reach, but always far from the resolution at which chemistry can be understood. Thus, structural and cell biology provide complementary, yet unconnected visions of the inner workings of cells. Here we discuss how the interplay between cryo-EM and cryo-electron tomography, as a connecting bridge to visualize macromolecules in situ, holds great promise to create comprehensive structural depictions of macromolecules as they interact in complex mixtures or, ultimately, inside the cell itself.
Asunto(s)
Biología Celular , Células , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Microscopía por Crioelectrón/métodos , Microscopía por Crioelectrón/tendencias , Tomografía con Microscopio Electrónico/métodos , Tomografía con Microscopio Electrónico/tendencias , Sustancias Macromoleculares/análisis , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Sustancias Macromoleculares/ultraestructura , Biología Celular/instrumentación , Células/química , Células/citología , Células/metabolismo , Células/ultraestructura , HumanosRESUMEN
Structural insights into macromolecular and protein complexes provide key clues about the molecular basis of the function. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful structural biology method for studying protein and macromolecular structures at high resolution in both native and near-native states. Despite the ability to get detailed structural insights into the processes underlying protein function using cryo-EM, there has been hesitancy amongst plant biologists to apply the method for biomolecular interaction studies. This is largely evident from the relatively fewer structural depositions of proteins and protein complexes from plant origin in electron microscopy databank. Even though the progress has been slow, cryo-EM has significantly contributed to our understanding of the molecular biology processes underlying photosynthesis, energy transfer in plants, besides viruses infecting plants. This chapter introduces sample preparation for both negative-staining electron microscopy (NSEM) and cryo-EM for plant proteins and macromolecular complexes and data analysis using single particle analysis for beginners.
Asunto(s)
Microscopía por Crioelectrón , Sustancias Macromoleculares , Microscopía por Crioelectrón/métodos , Sustancias Macromoleculares/ultraestructura , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/ultraestructura , Proteínas de Plantas/química , Coloración Negativa/métodosRESUMEN
Cryo-electron microscopy has become a powerful tool to determine three-dimensional (3D) structures of rigid biological macromolecules from noisy micrographs with single-particle reconstruction. Recently, deep neural networks, e.g., CryoDRGN, have demonstrated conformational and compositional heterogeneity of complexes. However, the lack of ground-truth conformations poses a challenge to assess the performance of heterogeneity analysis methods. In this work, variational autoencoders (VAE) with three types of deep generative priors were learned for latent variable inference and heterogeneous 3D reconstruction via Bayesian inference. More specifically, VAEs with "Variational Mixture of Posteriors" priors (VampPrior-SPR), non-parametric exemplar-based priors (ExemplarPrior-SPR) and priors from latent score-based generative models (LSGM-SPR) were quantitatively compared with CryoDRGN. We built four simulated datasets composed of hypothetical continuous conformation or discrete states of the hERG K + channel. Empirical and quantitative comparisons of inferred latent representations were performed with affine-transformation-based metrics. These models with more informative priors gave better regularized, interpretable factorized latent representations with better conserved pairwise distances, less deformed latent distributions and lower within-cluster variances. They were also tested on experimental datasets to resolve compositional and conformational heterogeneity (50S ribosome assembly, cowpea chlorotic mottle virus, and pre-catalytic spliceosome) with comparable high resolution. Codes and data are available: https://github.com/benjamin3344/DGP-SPR.
Asunto(s)
Teorema de Bayes , Microscopía por Crioelectrón , Imagenología Tridimensional , Microscopía por Crioelectrón/métodos , Imagenología Tridimensional/métodos , Algoritmos , Procesamiento de Imagen Asistido por Computador/métodos , Redes Neurales de la Computación , Sustancias Macromoleculares/química , Sustancias Macromoleculares/ultraestructuraRESUMEN
For decades, bacteria were thought of as "bags" of enzymes, lacking organelles and significant subcellular structures. This stood in sharp contrast with eukaryotes, where intracellular compartmentalization and the role of large-scale order had been known for a long time. However, the emerging field of Bacterial Cell Biology has established that bacteria are in fact highly organized, with most macromolecular components having specific subcellular locations that can change depending on the cell's physiological state (Barry & Gitai, 2011; Lenz & Søgaard-Andersen, 2011; Thanbichler & Shapiro, 2008). For example, we now know that many processes in bacteria are orchestrated by cytoskeletal proteins, which polymerize into surprisingly diverse superstructures, such as rings, sheets, and tread-milling rods (Pilhofer & Jensen, 2013). These superstructures connect individual proteins, macromolecular assemblies, and even two neighboring cells, to affect essential higher-order processes including cell division, DNA segregation, and motility. Understanding these processes requires resolving the in vivo dynamics and ultrastructure at different functional stages of the cell, at macromolecular resolution and in 3-dimensions (3D). Fluorescence light microscopy (fLM) of tagged proteins is highly valuable for investigating protein localization and dynamics, and the resolution power of transmission electron microscopy (TEM) is required to elucidate the structure of macromolecular complexes in vivo and in vitro. This chapter summarizes the most recent advances in LM and TEM approaches that have revolutionized our knowledge and understanding of the microbial world.
Asunto(s)
Bacterias , Orgánulos , Bacterias/genética , Sustancias Macromoleculares/ultraestructura , Microscopía Electrónica de Transmisión , Microscopía Fluorescente/métodosRESUMEN
Recent developments in both instrumentation and image analysis algorithms have allowed three-dimensional electron microscopy (3D-EM) to increase automated image collections through large tissue volumes using serial block-face scanning EM (SEM) and to achieve near-atomic resolution of macromolecular complexes using cryo-electron tomography (cryo-ET) and sub-tomogram averaging. In this review, we discuss applications of cryo-ET to cell biology research on plant and algal systems and the special opportunities they offer for understanding the organization of eukaryotic organelles with unprecedently resolution. However, one of the most challenging aspects for cryo-ET is sample preparation, especially for multicellular organisms. We also discuss correlative light and electron microscopy (CLEM) approaches that have been developed for ET at both room and cryogenic temperatures.
Asunto(s)
Microscopía por Crioelectrón/métodos , Cianobacterias/ultraestructura , Tomografía con Microscopio Electrónico/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Sustancias Macromoleculares/ultraestructura , Orgánulos/ultraestructuraRESUMEN
Integrative modeling enables structure determination of macromolecular complexes by combining data from multiple experimental sources such as X-ray crystallography, electron microscopy or cross-linking mass spectrometry. It is particularly useful for complexes not amenable to high-resolution electron microscopy-complexes that are flexible, heterogeneous or imaged in cells with cryo-electron tomography. We have recently developed an integrative modeling protocol that allowed us to model multi-megadalton complexes as large as the nuclear pore complex. Here, we describe the Assembline software package, which combines multiple programs and libraries with our own algorithms in a streamlined modeling pipeline. Assembline builds ensembles of models satisfying data from atomic structures or homology models, electron microscopy maps and other experimental data, and provides tools for their analysis. Compared with other methods, Assembline enables efficient sampling of conformational space through a multistep procedure, provides new modeling restraints and includes a unique configuration system for setting up the modeling project. Our protocol achieves exhaustive sampling in less than 100-1,000 CPU-hours even for complexes in the megadalton range. For larger complexes, resources available in institutional or public computer clusters are needed and sufficient to run the protocol. We also provide step-by-step instructions for preparing the input, running the core modeling steps and assessing modeling performance at any stage.
Asunto(s)
Biología Computacional/métodos , Sustancias Macromoleculares , Modelos Moleculares , Programas Informáticos , Cristalografía por Rayos X , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Sustancias Macromoleculares/ultraestructura , Espectrometría de Masas , Microscopía ElectrónicaRESUMEN
Protein-nucleic acid interactions are involved in various biological processes such as gene expression, replication, transcription, translation and packaging. The binding affinities of protein-DNA and protein-RNA complexes are important for elucidating the mechanism of protein-nucleic acid recognition. Although experimental data on binding affinity are reported abundantly in the literature, no well-curated database is currently available for protein-nucleic acid binding affinity. We have developed a database, ProNAB, which contains more than 20 000 experimental data for the binding affinities of protein-DNA and protein-RNA complexes. Each entry provides comprehensive information on sequence and structural features of a protein, nucleic acid and its complex, experimental conditions, thermodynamic parameters such as dissociation constant (Kd), binding free energy (ΔG) and change in binding free energy upon mutation (ΔΔG), and literature information. ProNAB is cross-linked with GenBank, UniProt, PDB, ProThermDB, PROSITE, DisProt and Pubmed. It provides a user-friendly web interface with options for search, display, sorting, visualization, download and upload the data. ProNAB is freely available at https://web.iitm.ac.in/bioinfo2/pronab/ and it has potential applications such as understanding the factors influencing the affinity, development of prediction tools, binding affinity change upon mutation and design complexes with the desired affinity.
Asunto(s)
Bases de Datos de Proteínas , Sustancias Macromoleculares/clasificación , Ácidos Nucleicos/genética , Proteínas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Sustancias Macromoleculares/química , Sustancias Macromoleculares/ultraestructura , Mutación/genética , Ácidos Nucleicos/ultraestructura , Unión Proteica/genética , Proteínas/clasificaciónRESUMEN
Imaging of biomolecules by ionizing radiation, such as electrons, causes radiation damage which introduces structural and compositional changes of the specimen. The total number of high-energy electrons per surface area that can be used for imaging in cryogenic electron microscopy (cryo-EM) is severely restricted due to radiation damage, resulting in low signal-to-noise ratios (SNR). High resolution details are dampened by the transfer function of the microscope and detector, and are the first to be lost as radiation damage alters the individual molecules which are presumed to be identical during averaging. As a consequence, radiation damage puts a limit on the particle size and sample heterogeneity with which electron microscopy (EM) can deal. Since a transmission EM (TEM) image is formed from the scattering process of the electron by the specimen interaction potential, radiation damage is inevitable. However, we can aim to maximize the information transfer for a given dose and increase the SNR by finding alternatives to the conventional phase-contrast cryo-EM techniques. Here some alternative transmission electron microscopy techniques are reviewed, including phase plate, multi-pass transmission electron microscopy, off-axis holography, ptychography and a quantum sorter. Their prospects for providing more or complementary structural information within the limited lifetime of the sample are discussed.
Asunto(s)
Microscopía por Crioelectrón/métodos , Sustancias Macromoleculares/ultraestructura , Electrones , Relación Señal-Ruido , Relación Estructura-ActividadRESUMEN
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. We present here a more sophisticated way of adjusting the volumes before comparing, which implies adjustment of grey level scale and spectrum energy, but keeping phases intact inside a mask and imposing the results to be strictly positive. The adjustment that we propose leaves the volumes in the same numeric frame, allowing to perform operations among the adjusted volumes in a more reliable way. This adjustment can be a preliminary step for several applications such as comparison through subtraction, map sharpening, or combination of volumes through a consensus that selects the best resolved parts of each input map. Our development might also be used as a sharpening method using an atomic model as a reference. We illustrate the applicability of this algorithm with the reconstructions derived of several experimental examples. This algorithm is implemented in Xmipp software package and its applications are user-friendly accessible through the cryo-EM image processing framework Scipion.
Asunto(s)
Algoritmos , Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Sustancias Macromoleculares/ultraestructura , Cápside/química , Cápside/ultraestructura , Virus de la Hepatitis B/ultraestructura , Sustancias Macromoleculares/química , Modelos Moleculares , Conformación Molecular , Conformación Proteica , Reproducibilidad de los Resultados , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/ultraestructuraRESUMEN
Activator proteins 1 (AP-1) comprise one of the largest families of eukaryotic basic leucine zipper transcription factors. Despite advances in the characterization of AP-1 DNA-binding sites, our ability to predict new binding sites and explain how the proteins achieve different gene expression levels remains limited. Here we address the role of sequence-specific DNA flexibility for stability and specific binding of AP-1 factors, using microsecond-long molecular dynamics simulations. As a model system, we employ yeast AP-1 factor Yap1 binding to three different response elements from two genetic environments. Our data show that Yap1 actively exploits the sequence-specific flexibility of DNA within the response element to form stable protein-DNA complexes. The stability also depends on the four to six flanking nucleotides, adjacent to the response elements. The flanking sequences modulate the conformational adaptability of the response element, making it more shape-efficient to form specific contacts with the protein. Bioinformatics analysis of differential expression of the studied genes supports our conclusions: the stability of Yap1-DNA complexes, modulated by the flanking environment, influences the gene expression levels. Our results provide new insights into mechanisms of protein-DNA recognition and the biological regulation of gene expression levels in eukaryotes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , ADN/genética , Proteínas de Saccharomyces cerevisiae/genética , Factor de Transcripción AP-1/genética , Factores de Transcripción/genética , Secuencia de Bases/genética , Sitios de Unión/genética , ADN/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Regulación de la Expresión Génica/genética , Sustancias Macromoleculares/ultraestructura , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/ultraestructura , Simulación de Dinámica Molecular , Elementos de Respuesta/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Factor de Transcripción AP-1/ultraestructura , Factores de Transcripción/ultraestructura , Proteínas Señalizadoras YAPRESUMEN
The Y-family DNA polymerase η (Polη) is critical for the synthesis past damaged DNA nucleotides in yeast through translesion DNA synthesis (TLS). TLS is initiated by monoubiquitination of proliferating cell nuclear antigen (PCNA) and the subsequent recruitment of TLS polymerases. Although individual structures of the Polη catalytic core and PCNA have been solved, a high-resolution structure of the complex of Polη/PCNA or Polη/monoubiquitinated PCNA (Ub-PCNA) still remains elusive, partly due to the disordered Polη C-terminal region and the flexibility of ubiquitin on PCNA. To circumvent these obstacles and obtain structural insights into this important TLS polymerase complex, we developed photo-activatable PCNA and Ub-PCNA probes containing a p-benzoyl-L-phenylalanine (pBpa) crosslinker at selected positions on PCNA. By photo-crosslinking the probes with full-length Polη, specific crosslinking sites were identified following tryptic digestion and tandem mass spectrometry analysis. We discovered direct interactions of the Polη catalytic core and its C-terminal region with both sides of the PCNA ring. Model building using the crosslinking site information as a restraint revealed multiple conformations of Polη in the polymerase complex. Availability of the photo-activatable PCNA and Ub-PCNA probes will also facilitate investigations into other PCNA-containing complexes important for DNA replication, repair and damage tolerance.
Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , ADN/biosíntesis , Antígeno Nuclear de Célula en Proliferación/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/genética , Benzofenonas/farmacología , ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/ultraestructura , Sustancias Macromoleculares/química , Sustancias Macromoleculares/ultraestructura , Mutación/genética , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/ultraestructura , Unión Proteica/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura , Ubiquitina/química , Ubiquitina/ultraestructuraRESUMEN
The electrochemical behavior and the interaction of the immunosuppressive drug azathioprine (AZA) with deoxyribonucleic acid (DNA) were investigated using voltammetric techniques, mass spectrometry (MS), and scanning electron microscopy (SEM). The redox mechanism of AZA on glassy carbon (GC) was investigated using cyclic and differential pulse (DP) voltammetry. It was proven that the electroactive center of AZA is the nitro group and its reduction mechanism is a diffusion-controlled process, which occurs in consecutive steps with formation of electroactive products and involves the transfer of electrons and protons. A redox mechanism was proposed and the interaction of AZA with DNA was also investigated. Morphological characterization of the DNA film on the electrode surface before and after interaction with AZA was performed using scanning electron microscopy. An electrochemical DNA biosensor was employed to study the interactions between AZA and DNA with different concentrations, incubation times, and applied potential values. It was shown that the reduction of AZA molecules bound to the DNA layer induces structural changes of the DNA double strands and oxidative damage, which were recognized through the occurrence of the 8-oxo-deoxyguanosine oxidation peak. Mass spectrometry investigation of the DNA film before and after interaction with AZA also demonstrated the formation of AZA adducts with purine bases.
Asunto(s)
Azatioprina/química , Azatioprina/metabolismo , ADN/química , ADN/metabolismo , Oxidación-Reducción , Algoritmos , Azatioprina/farmacología , Técnicas Biosensibles , Fenómenos Químicos , Sustancias Macromoleculares/química , Sustancias Macromoleculares/ultraestructura , Espectrometría de Masas , Modelos TeóricosRESUMEN
Over the last few years, cryo electron microscopy has become the most important method in structural biology. While 80% of deposited maps are from single particle analysis, electron tomography has grown to become the second most important method. In particular sub-tomogram averaging has matured as a method, delivering structures between 2 and 5 Å from complexes in cells as well as in vitro complexes. While this resolution range is not standard, novel developments point toward a promising future. Here, we provide a guide for the workflow from sample to structure to gain insight into this emerging field.
Asunto(s)
Biología/métodos , Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Sustancias Macromoleculares/ultraestructura , Orgánulos/ultraestructura , Imagen Individual de Molécula/métodos , Animales , Biología/tendencias , HumanosRESUMEN
For a more complete understanding of molecular mechanisms, it is important to study macromolecules and their assemblies in the broader context of the cell. This context can be visualized at nanometer resolution in three dimensions (3D) using electron cryo-tomography, which requires tilt series to be recorded and computationally aligned, currently limiting throughput. Additionally, the high-resolution signal preserved in the raw tomograms is currently limited by a number of technical difficulties, leading to an increased false-positive detection rate when using 3D template matching to find molecular complexes in tomograms. We have recently described a 2D template matching approach that addresses these issues by including high-resolution signal preserved in single-tilt images. A current limitation of this approach is the high computational cost that limits throughput. We describe here a GPU-accelerated implementation of 2D template matching in the image processing software cisTEM that allows for easy scaling and improves the accessibility of this approach. We apply 2D template matching to identify ribosomes in images of frozen-hydrated Mycoplasma pneumoniae cells with high precision and sensitivity, demonstrating that this is a versatile tool for in situ visual proteomics and in situ structure determination. We benchmark the results with 3D template matching of tomograms acquired on identical sample locations and identify strengths and weaknesses of both techniques, which offer complementary information about target localization and identity.
Asunto(s)
Células/química , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Sustancias Macromoleculares/química , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Sustancias Macromoleculares/ultraestructura , Mycoplasma pneumoniae/química , Proteómica/métodos , Ribosomas/química , Programas InformáticosRESUMEN
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.
Asunto(s)
Microscopía por Crioelectrón/métodos , Sustancias Macromoleculares/química , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Proteínas/química , Humanos , Aprendizaje Automático , Sustancias Macromoleculares/metabolismo , Sustancias Macromoleculares/ultraestructura , Modelos Moleculares , Redes Neurales de la Computación , Conformación Proteica , Multimerización de Proteína , Proteínas/metabolismo , Proteínas/ultraestructura , Máquina de Vectores de Soporte , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/ultraestructuraRESUMEN
The advances in electron cryo-microscopy have enabled high-resolution structural studies of vitrified macromolecular complexes in situ by cryo-electron tomography (cryo-ET). Since utilization of cryo-ET is generally limited to the specimens with thickness < 500 nm, a complex sample preparation protocol to study larger samples such as single eukaryotic cells by cryo-ET was developed and optimized over the last decade. The workflow is based on the preparation of a thin cellular lamella by cryo-focused ion beam milling (cryo-FIBM) from the vitrified cells. The sample preparation protocol is a multi-step process which includes utilization of several high-end instruments and comprises sample manipulation prone to sample deterioration. Here, we present a workflow for preparation of three different model specimens that was optimized to provide high-quality lamellae for cryo-ET or electron diffraction tomography with high reproducibility. Preparation of lamellae from large adherent mammalian cells, small suspension eukaryotic cell line, and protein crystals of intermediate size is described which represents examples of the most frequently studied samples used for cryo-FIBM in life sciences.
Asunto(s)
Microscopía por Crioelectrón/métodos , Sustancias Macromoleculares/ultraestructura , Manejo de Especímenes/métodos , Animales , Células/ultraestructura , Tomografía con Microscopio Electrónico/métodos , Iones , Biología Molecular/métodos , Proteínas/ultraestructura , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/ultraestructura , Flujo de TrabajoRESUMEN
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. In this chapter, we illustrate this whole processing pipeline putting in value the strength of "meta algorithms," which are the combination of several algorithms, each one with different mathematical rationale, in order to distinguish correctly from incorrectly estimated parameters. We show how this strategy leads to superior performance of the whole pipeline as well as more confident assessments about the reconstructed structures. The "meta algorithms" strategy is common to many fields and, in particular, it has provided excellent results in bioinformatics. We illustrate this combination using the workflow engine, Scipion.
Asunto(s)
Algoritmos , Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Imagen Individual de Molécula/métodos , Biología Computacional , Sustancias Macromoleculares/ultraestructura , Biología Molecular/métodos , Flujo de TrabajoRESUMEN
Macromolecular crystallography (MX) leverages the methods of physics and the language of chemistry to reveal fundamental insights into biology. Often beautifully artistic images present MX results to support profound functional hypotheses that are vital to entire life science research community. Over the past several decades, synchrotrons around the world have been the workhorses for X-ray diffraction data collection at many highly automated beamlines. The newest tools include X-ray-free electron lasers (XFELs) located at facilities in the USA, Japan, Korea, Switzerland, and Germany that deliver about nine orders of magnitude higher brightness in discrete femtosecond long pulses. At each of these facilities, new serial femtosecond crystallography (SFX) strategies exploit slurries of micron-size crystals by rapidly delivering individual crystals into the XFEL X-ray interaction region, from which one diffraction pattern is collected per crystal before it is destroyed by the intense X-ray pulse. Relatively simple adaptions to SFX methods produce time-resolved data collection strategies wherein reactions are triggered by visible light illumination or by chemical diffusion/mixing. Thus, XFELs provide new opportunities for high temporal and spatial resolution studies of systems engaged in function at physiological temperature. In this chapter, we summarize various issues related to microcrystal slurry preparation, sample delivery into the X-ray interaction region, and some emerging strategies for time-resolved SFX data collection.
Asunto(s)
Cristalografía por Rayos X/métodos , Rayos Láser , Sustancias Macromoleculares/química , Sincrotrones , Difracción de Rayos X/métodos , Cristalografía por Rayos X/instrumentación , Electrones , Sustancias Macromoleculares/ultraestructura , Biología Molecular , Proteínas/química , Proteínas/ultraestructura , Difracción de Rayos X/instrumentaciónRESUMEN
Single molecule localization microscopy offers in principle resolution down to the molecular level, but in practice this is limited primarily by incomplete fluorescent labeling of the structure. This missing information can be completed by merging information from many structurally identical particles. In this work, we present an approach for 3D single particle analysis in localization microscopy which hugely increases signal-to-noise ratio and resolution and enables determining the symmetry groups of macromolecular complexes. Our method does not require a structural template, and handles anisotropic localization uncertainties. We demonstrate 3D reconstructions of DNA-origami tetrahedrons, Nup96 and Nup107 subcomplexes of the nuclear pore complex acquired using multiple single molecule localization microscopy techniques, with their structural symmetry deducted from the data.
Asunto(s)
Sustancias Macromoleculares/química , Sustancias Macromoleculares/ultraestructura , Imagen Individual de Molécula/métodos , Algoritmos , Línea Celular , Simulación por Computador , ADN/química , ADN/ultraestructura , Humanos , Imagenología Tridimensional , Conformación Molecular , Poro Nuclear/química , Poro Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/ultraestructura , Relación Señal-Ruido , Imagen Individual de Molécula/estadística & datos numéricosRESUMEN
Two light-activated NO donors [RuCl(qn)(Lbpy)(NO)]X with 8-hydroxyquinoline (qn) and 2,2'-bipyridine derivatives (Lbpy) as co-ligands were synthesized (Lbpy1 = 4,4'-dicarboxyl-2,2'-dipyridine, X = Cl- and Lbpy2 = 4,4'-dimethoxycarbonyl-2,2'-dipyridine, X = NO3-), and characterized using ultraviolet-visible (UV-vis) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (1H NMR), elemental analysis and electrospray ionization mass spectrometry (ESI-MS) spectra. The [RuCl(qn)(Lbpy2)(NO)]NO3 complex was crystallized and exhibited distorted octahedral geometry, in which the Ru-N(O) bond length was 1.752(6) Å and the Ru-N-O angle was 177.6(6)°. Time-resolved FT-IR and electron paramagnetic resonance (EPR) spectra were used to confirm the photoactivated NO release of the complexes. The binding constant (Kb) of two complexes with human serum albumin (HSA) and DNA were quantitatively evaluated using fluorescence spectroscopy, Ru-Lbpy1 (Kb~106 with HSA and ~104 with DNA) had higher affinity than Ru-Lbpy2. The interactions between the complexes and HSA were investigated using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and EPR spectra. HSA can be used as a carrier to facilitate the release of NO from the complexes upon photoirradiation. The confocal imaging of photo-induced NO release in living cells was successfully observed with a fluorescent NO probe. Moreover, the photocleavage of pBR322 DNA for the complexes and the effect of different Lbpy substituted groups in the complexes on their reactivity were analyzed.