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Simple and robust 3D MINFLUX excitation with a variable phase plate

Light(Science & Applications) 2024年第7期13卷 1354-1363页

作者： Takahiro Deguchi Jonas Ries European Molecular Biology Laboratory Cell Biology and BiophysicsHeidelbergGermany. Max Perutz Labs Vienna Biocenter Campus(VBC)ViennaAustria. University of Vienna Center for Molecular BiologyDepartment of Structural and Computational BiologyViennaAustria. University of Vienna Faculty of PhysicsViennaAustria

MINFLUX has achieved extraordinary resolution in superresolution imaging and single fluorophore *** is based on localizing single fluorophores by rapid probing with a patterned beam that features a local intensity *** implementations,however,are complex and expensive and are limited in speed and ***,we show that a combination of an electro-optical modulator with a segmented birefringent element such as a spatial light modulator produces a variable phase plate for which the phase can be scanned on the MHz *** or top-hat phase patterns generate high-contrast compact excitation point-spread functions for MINFLUX localization in the x,y,and z-direction,respectively,which can be scanned across a fluorophore within a microsecond,switched within 60 microseconds and alternated among different excitation *** discuss how to compensate for non-optimal performance of the components and present a robust 3D and multi-color MINFLUX excitation module,which we envision as an integral component of a high-performance and cost-effective open-source MINFLUX.

关键词： excitation phase switched

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Dormancy heterogeneity among Arabidopsis thaliana seeds is linked to individual seed size

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Plant Communications 2024年第2期5卷 215-227页

作者： Michal Krzyszton Sebastian P.Sacharowski Veena Halale Manjunath Katarzyna Muter Grzegorz Bokota Ce Wang Dariusz Plewczynski Tereza Dobisova Szymon Swiezewski Laboratory of Seeds Molecular Biology Institute of Biochemistry and BiophysicsPAS02-106 WarsawPoland Laboratory of Functional and Structural Genomics Centre of New TechnologiesUniversity of WarsawWarsawPoland Laboratory of Bioinformatics and Computational Genomics Faculty of Mathematics and Information ScienceWarsaw University of TechnologyWarsawPoland Labdeers Ltd. 680-01 BoskoviceCzech Republic

Production of morphologically and physiologically variable seeds is an important strategy that helps plants to survive in unpredictable natural ***,the model plant Arabidopsis thaliana and most agronomically essential crops produce visually homogenous *** automated phenotype analysis,we observed that small seeds in Arabidopsis tend to have higher primary and secondary dormancy levels than large *** analysis revealed distinct gene expression profiles between large and small *** seeds have higher expression of translation-related genes implicated in germination *** contrast,small seeds have elevated expression of many positive regulators of dormancy,including a key regulator of this process,the DOG1 *** in DOG1 expression are associated with differential production of its alternative cleavage and polyadenylation isoforms;in small seeds,the proximal poly(A)site is selected,resulting in a short mRNA ***,single-seed RNA sequencing analysis demonstrated that large seeds resemble DOG1 knockout mutant ***,on the single-seed level,expression of genes affected by seed size is correlated with expression of genes that position seeds on the path toward *** results demonstrate an unexpected link between seed size and dormancy phenotypes in a species that produces highly homogenous seed pools,suggesting that the correlation between seed morphology and physiology is more widespread than initially assumed.

关键词： seed size dormancy DOG1 Arabidopsis

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Approximations of MINFLUX Localization Precision with Background

arXiv

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arXiv 2024年

作者： Marin, Zach Ries, Jonas Vienna Austria University of Vienna Center for Molecular Biology Department of Structural and Computational Biology Vienna Austria European Molecular Biology Laboratory Cell Biology and Biophysics Heidelberg Germany University of Vienna Faculty of Physics Vienna Austria

MINFLUX [1] is an emerging super-resolution technology that measures the position of single fluorophores with nanometer precision using fewer photons than any other fluorescence imaging or tracking technique. Here, we derive simple and instructive analytical equations for MINFLUX localization precision with a special focus on background photons. A fluorescence background, either arising from an imperfect zero of the MINFLUX excitation point spread function (PSF) or from auto- or out-of-focus fluorescence, ultimately limits the resolution achievable with MINFLUX. Building on previous work [1–3], we try to improve our understanding of the influence of background, especially when it is unknown, through a new set of expressions for the localization precision, based on an explicit background term instead of signal-to-background ratio. We use these equations to generate an intuitive understanding of how fluorescence background affects MINFLUX measurements and illustrate that: • The precision of an emitter position estimate depends on the gradient of its excitation profile. • Knowledge of the fluorescence background, obtained during post-processing of MINFLUX traces or through separate measurements, provides a better localization precision than in the case of unknown background. • In diffraction-limited systems, localization with a PSF that features a near-zero minimum outperforms localization with a maximum. We also present an analytical expression for the localization precision in orbital tracking [4], which we use for comparison to MINFLUX. Non-trivial derivations presented in this paper can be found in the accompanying Mathematica notebook. © 2024, CC BY.

关键词： Optical transfer function

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MINFLUX - molecular resolution with minimal photons

arXiv

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arXiv 2024年

作者： Scheiderer, Lukas Marin, Zach Ries, Jonas Vienna Austria University of Vienna Center for Molecular Biology Department of Structural and Computational Biology Vienna Austria European Molecular Biology Laboratory Cell Biology and Biophysics Heidelberg Germany University of Vienna Faculty of Physics Vienna Austria

Optical super-resolution microscopy is a key technology for structural biology that optimally complements electron microscopy because of its high contrast and live-cell compatibility. MINFLUX is an emerging super-resolution technique that measures the precise position of single fluorophores by targeted scanning of a patterned excitation beam featuring a minimum. It achieves unprecedented localization precision for a given number of detected photons. In combination with switchable fluorophores, it can be used for super-resolution imaging with nanometer resolution. For tracking of single fluorophores, it greatly outperforms camera-based techniques and achieves nanometer spatial and sub-millisecond temporal resolution over long tracks. Here, we introduce the basic principle of MINFLUX and explain how it can reach such high photon efficiencies. We then discuss the potential and limitations of MINFLUX for superresolution imaging and single-fluorophore tracking, describe recent extensions and variations of MINFLUX and give an outlook to future developments. © 2024, CC BY.

关键词： Scanning electron microscopy

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Predicting dynamic cellular protein-RNA interactions by deep learning using in vivo RNA structures

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细胞研究（英文版） 2021年第5期31卷 495-516页

作者： Lei Sun Kui Xu Wenze Huang Yucheng T.Yang Pan Li Lei Tang Tuanlin Xiong Qiangfeng Cliff Zhang MOE Key Laboratory of Bioinformatics Beijing Advanced Innovation Center for Structural Biology and Frontier Research Center for Biological StructureCenter for Synthetic and Systems BiologySchool of Life SciencesTsinghua UniversityBeijing 100084China Tsinghua-Peking Center for Life Sciences Beijing 100084China Program in Computational Biology and Bioinformatics Yale UniversityNew HavenCT 06520USA Department of Molecular Biophysics and Biochemistry Yale UniversityNew HavenCT 06520USA

Interactions with RNA-binding proteins (RBPs) are integral to RNA function and cellular regulation,and dynamically reflect specific cellular ***,presently available tools for predicting RBP-RNA interactions employ RNA sequence and/or predicted RNA structures,and therefore do not capture their condition-dependent ***,after profiling transcriptome-wide in vivo RNA secondary structures in seven cell types,we developed PrismNet,a deep learning tool that integrates experimental in vivo RNA structure data and RBP binding data for matched cells to accurately predict dynamic RBP binding in various cellular *** results for 168 RBPs support its utility for both understanding CUP-seq results and largely extending such interaction data to accurately analyze additional cell ***,PrismNet employs an 'attention' strategy to computationally identify exact RBP-binding nucleotides,and we discovered enrichment among dynamic RBP-binding sites for structure-changing variants(riboSNitches),which can link genetic diseases with dysregulated RBP *** rich profiling data and deep learning-based prediction tool provide access to a previously inaccessible layer of cell-type-specific RBP-RNA interactions,with clear utility for understanding and treating human diseases.

关键词： Bioinformatics Mechanisms of disease molecular biology

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Higher-Order Generalization Bounds: Learning Deep Probabilistic programs via PAC-Bayes Objectives

arXiv

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arXiv 2022年

作者： Warrell, Jonathan Gerstein, Mark Program in Computational Biology and Bioinformatics Department of Molecular Biophysics and Biochemistry Yale University New HavenCT06520 United States

Deep Probabilistic programming (DPP) allows powerful models based on recursive computation to be learned using efficient deep-learning optimization techniques. Additionally, DPP offers a unified perspective, where inference and learning algorithms are treated on a par with models as stochastic programs. Here, we offer a framework for representing and learning flexible PAC-Bayes bounds as stochastic programs using DPP-based methods. In particular, we show that DPP techniques may be leveraged to derive generalization bounds that draw on the compositionality of DPP representations. In turn, the bounds we introduce offer principled training objectives for higher-order probabilistic programs. We offer a definition of a higher-order generalization bound, which naturally encompasses single- and multi-task generalization perspectives (including transfer- and meta-learning) and a novel class of bound based on a learned measure of model complexity. Further, we show how modified forms of all higher-order bounds can be efficiently optimized as objectives for DPP training, using variational techniques. We test our framework using single- and multi-task generalization settings on synthetic and biological data, showing improved performance and generalization prediction using flexible DPP model representations and learned complexity measures. Copyright © 2022, The Authors. All rights reserved.

关键词： Deep learning

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The condensin holocomplex cycles dynamically between open and collapsed states (Sep, 10.1038/s41594-020-0508-3, 2020)

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NATURE structural & molecular biology 2020年第12期27卷 1211-1211页

作者： Ryu, Je-Kyung Katan, Allard J. van der Sluis, Eli O. Wisse, Thomas de Groot, Ralph Haering, Christian H. Dekker, Cees Department of Bionanoscience Kavli Institute of Nanoscience Delft Delft University of Technology Delft the Netherlands Cell Biology and Biophysics Unit Structural and Computational Unit European Molecular Biology Laboratory (EMBL) Heidelberg Germany

structural maintenance of chromosome (SMC) protein complexes are the key organizers of the spatiotemporal structure of chromosomes. The condensin SMC complex has recently been shown to be a molecular motor that extrudes large loops of DNA, but the mechanism of this unique motor remains elusive. Using atomic force microscopy, we show that budding yeast condensin exhibits mainly open ‘O’ shapes and collapsed ‘B’ shapes, and it cycles dynamically between these two states over time, with ATP binding inducing the O to B transition. Condensin binds DNA via its globular domain and also via the hinge domain. We observe a single condensin complex at the stem of extruded DNA loops, where the neck size of the DNA loop correlates with the width of the condensin complex. The results are indicative of a type of scrunching model in which condensin extrudes DNA by a cyclic switching of its conformation between O and B shapes.

关键词： Atomic force microscopy Nuclear organization Single-molecule biophysics

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Correction to: Efficient genetic code expansion without host genome modifications (Nature Biotechnology, (2024), 10.1038/s41587-024-02385-y)

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Nature Biotechnology 2024年

作者： Costello, Alan Peterson, Alexander A. Lanster, David L. Li, Zhiyi Carver, Gavriela D. Badran, Ahmed H. Department of Chemistry The Scripps Research Institute La Jolla CA United States Department of Integrative Structural and Computational Biology The Scripps Research Institute La Jolla CA United States Doctoral Program in Chemical and Biological Sciences The Scripps Research Institute La Jolla CA United States Department of Molecular Biology Princeton University PrincetonNJ United States

Correction to: Nature Biotechnologyhttps://***/10.1038/s41587-024-02385-y, published online 11 September 2024. In the version of the article initially published, the far left oxygen symbol was missing from Fig. 1b and "N346A;C348A" was mistakenly included in the figure legend for Fig. 4d. These errors have now been amended in the HTML and PDF versions of the article. © The Author(s), under exclusive licence to Springer Nature America, Inc. 2024.

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structural insights into the role of N-terminal integrity in PhoSL for core-fucosylated N-glycan recognition

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International Journal of Biological Macromolecules 2024年 255卷 128309页

作者： Lou, Yuan-Chao Tu, Cheng-Fen Chou, Chun-Chi Yeh, Hsin-Hong Chien, Chia-Yu Sadotra, Sushant Chen, Chinpan Yang, Ruey-Bing Hsu, Chun-Hua Institute of Biomedical Sciences Academia Sinica Taipei115 Taiwan Department of Agricultural Chemistry National Taiwan University Taipei106 Taiwan Chemical Biology and Molecular Biophysics Taiwan International Graduate Program Academia Sinica Taipei115 Taiwan Institute of Bioinformatics and Structural Biology National Tsing Hua University Hsinchu300 Taiwan Biomedical Translation Research Center Academia Sinica Taipei115 Taiwan Ph.D. Program in Drug Discovery and Development Industry College of Pharmacy Taipei Medical University Taipei110 Taiwan Genome and Systems Biology Degree Program National Taiwan University and Academia Sinica Taipei106 Taiwan Institute of Biochemical Sciences National Taiwan University Taipei106 Taiwan Center for Computational and Systems Biology National Taiwan University Taipei106 Taiwan

PhoSL (Pholiota squarrosa Lectin) has an exceptional binding affinity for biomolecules with core-fucosylated N-glycans. This modification involves the addition of fucose to the inner N-acetylglucosamine within the N-glycan structure and is known to influence many physiological processes. Nevertheless, the molecular interactions underlying high-affinity binding of native PhoSL to core-fucosylated N-glycans remain largely unknown. In this study, we devised a strategy to produce PhoSL with the essential structural characteristics of the native protein (n-PhoSL). To do so, a fusion protein was expressed in E. coli and purified. Then, enzymatic cleavage and incubation with glutathione were utilized to recapitulate the native primary structure and disulfide bonding pattern. Subsequently, we identified the residues crucial for n-PhoSL binding to core-fucosylated chitobiose (N2F) via NMR spectroscopy. Additionally, crystal structures were solved for both apo n-PhoSL and its N2F complex. These analyses suggested a pivotal role of the N-terminal amine in maintaining the integrity of the binding pocket and actively contributing to core-fucose recognition. In support of this idea, the inclusion of additional residues at the N-terminus considerably reduced binding affinity and PhoSL cytotoxicity toward breast cancer cells. Taken together, these findings can facilitate the utilization of PhoSL in basic research, diagnostics and therapeutic strategies. © 2023 Elsevier B.V.

关键词： Proteins

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Graph-based information diffusion method for prioritizing functionally related genes in protein-protein interaction networks 25

Graph-based information diffusion method for prioritizing fu...

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25th Pacific Symposium on Biocomputing, PSB 2020

作者： Pham, Minh Lichtarge, Olivier Integrative Molecular and Biomedical Sciences Graduate Program Department of Molecular and Human Genetics Baylor College of Medicine One Baylor Plaza HoustonTX77030 United States Departments of Molecular and Human Genetics Structural and Computational Biology and Molecular Biophysics Biochemistry and Molecular Biology and Pharmacology One Baylor Plaza HoustonTX77030 United States

Shortest path length methods are routinely used to validate whether genes of interest are functionally related to each other based on biological network information. However, the methods are computationally intensive, impeding extensive utilization of network information. In addition, nonweighted shortest path length approach, which is more frequently used, often treat all network connections equally without taking into account of confidence levels of the associations. On the other hand, graph-based information diffusion method, which employs both the presence and confidence weights of network edges, can efficiently explore large networks and has previously detected meaningful biological patterns. Therefore, in this study, we hypothesized that the graph-based information diffusion method could prioritize genes with relevant functions more efficiently and accurately than the shortest path length approaches. We demonstrated that the graph-based information diffusion method substantially differentiated not only genes participating in same biological pathways (p 0.0001) but also genes associated with specific human drug-induced clinical symptoms (p 0.0001) from random. Furthermore, the diffusion method prioritized these functionally related genes faster and more accurately than the shortest path length approaches (pathways: p = 2.7e-28, clinical symptoms: p = 0.032). These data show the graph-based information diffusion method can be routinely used for robust prioritization of functionally related genes, facilitating efficient network validation and hypothesis generation, especially for human phenotypespecific genes. © 2019 The Authors.

关键词： Genes

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