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Nature Methods

Björn Hellenkamp, Philipp Wortmann, Florian Kandzia, Martin Zacharias, Thorsten Hugel
We present an approach that enables us to simultaneously access structure and dynamics of a multidomain protein in solution. Dynamic domain arrangements are experimentally determined by combining self-consistent networks of distance distributions with known domain structures. Local structural dynamics are correlated with the global arrangements by analyzing networks of time-resolved single-molecule fluorescence parameters. The strength of this hybrid approach is shown by an application to the flexible multidomain protein Hsp90...
December 5, 2016: Nature Methods
Friedhelm Serwane, Alessandro Mongera, Payam Rowghanian, David A Kealhofer, Adam A Lucio, Zachary M Hockenbery, Otger Campàs
The mechanical properties of the cellular microenvironment and their spatiotemporal variations are thought to play a central role in sculpting embryonic tissues, maintaining organ architecture and controlling cell behavior, including cell differentiation. However, no direct in vivo and in situ measurement of mechanical properties within developing 3D tissues and organs has yet been performed. Here we introduce a technique that employs biocompatible, magnetically responsive ferrofluid microdroplets as local mechanical actuators and allows quantitative spatiotemporal measurements of mechanical properties in vivo...
December 5, 2016: Nature Methods
Reza Kalhor, Prashant Mali, George M Church
We present an approach for engineering evolving DNA barcodes in living cells. A homing guide RNA (hgRNA) scaffold directs the Cas9-hgRNA complex to the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show that they can be used to record lineage history and that the barcode RNA can be amplified in situ, a prerequisite for in situ sequencing...
December 5, 2016: Nature Methods
Zechen Chong, Jue Ruan, Min Gao, Wanding Zhou, Tenghui Chen, Xian Fan, Li Ding, Anna Y Lee, Paul Boutros, Junjie Chen, Ken Chen
We present novoBreak, a genome-wide local assembly algorithm that discovers somatic and germline structural variation breakpoints in whole-genome sequencing data. novoBreak consistently outperformed existing algorithms on real cancer genome data and on synthetic tumors in the ICGC-TCGA DREAM 8.5 Somatic Mutation Calling Challenge primarily because it more effectively utilized reads spanning breakpoints. novoBreak also demonstrated great sensitivity in identifying short insertions and deletions.
November 28, 2016: Nature Methods
Taibo Li, Rasmus Wernersson, Rasmus B Hansen, Heiko Horn, Johnathan Mercer, Greg Slodkowicz, Christopher T Workman, Olga Rigina, Kristoffer Rapacki, Hans H Stærfeldt, Søren Brunak, Thomas S Jensen, Kasper Lage
Genome-scale human protein-protein interaction networks are critical to understanding cell biology and interpreting genomic data, but challenging to produce experimentally. Through data integration and quality control, we provide a scored human protein-protein interaction network (InWeb_InBioMap, or InWeb_IM) with severalfold more interactions (>500,000) and better functional biological relevance than comparable resources. We illustrate that InWeb_InBioMap enables functional interpretation of >4,700 cancer genomes and genes involved in autism...
November 28, 2016: Nature Methods
Daphne S Bindels, Lindsay Haarbosch, Laura van Weeren, Marten Postma, Katrin E Wiese, Marieke Mastop, Sylvain Aumonier, Guillaume Gotthard, Antoine Royant, Mark A Hink, Theodorus W J Gadella
We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red Förster proteins as a fusion tag, and it is especially useful as a fluorescence resonance energy transfer (FRET) acceptor in ratiometric imaging...
November 21, 2016: Nature Methods
Yashar S Niknafs, Balaji Pandian, Hariharan K Iyer, Arul M Chinnaiyan, Matthew K Iyer
Accurate transcript structure and abundance inference from RNA sequencing (RNA-seq) data is foundational for molecular discovery. Here we present TACO, a computational method to reconstruct a consensus transcriptome from multiple RNA-seq data sets. TACO employs novel change-point detection to demarcate transcript start and end sites, leading to improved reconstruction accuracy compared with other tools in its class. The tool is available at and can be readily incorporated into RNA-seq analysis workflows...
November 21, 2016: Nature Methods
Christian Franke, Markus Sauer, Sebastian van de Linde
We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization...
November 21, 2016: Nature Methods
Mahmoud L Nasr, Diego Baptista, Mike Strauss, Zhen-Yu J Sun, Simina Grigoriu, Sonja Huser, Andreas Plückthun, Franz Hagn, Thomas Walz, James M Hogle, Gerhard Wagner
We engineered covalently circularized nanodiscs (cNDs) which, compared with standard nanodiscs, exhibit enhanced stability, defined diameter sizes and tunable shapes. Reconstitution into cNDs enhanced the quality of nuclear magnetic resonance spectra for both VDAC-1, a β-barrel membrane protein, and the G-protein-coupled receptor NTR1, an α-helical membrane protein. In addition, we used cNDs to visualize how simple, nonenveloped viruses translocate their genomes across membranes to initiate infection.
November 21, 2016: Nature Methods
Mario Kompauer, Sven Heiles, Bernhard Spengler
We report an atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) setup with a lateral resolution of 1.4 μm, a mass resolution greater than 100,000, and accuracy below ±2 p.p.m. We achieved this by coupling a focusing objective with a numerical aperture (NA) of 0.9 at 337 nm and a free working distance of 18 mm in coaxial geometry to an orbitrap mass spectrometer and optimizing the matrix application. We demonstrate improvement in image contrast, lateral resolution, and ion yield per unit area compared with a state-of-the-art commercial MSI source...
November 14, 2016: Nature Methods
Andrew Palmer, Prasad Phapale, Ilya Chernyavsky, Regis Lavigne, Dominik Fay, Artem Tarasov, Vitaly Kovalev, Jens Fuchser, Sergey Nikolenko, Charles Pineau, Michael Becker, Theodore Alexandrov
High-mass-resolution imaging mass spectrometry promises to localize hundreds of metabolites in tissues, cell cultures, and agar plates with cellular resolution, but it is hampered by the lack of bioinformatics tools for automated metabolite identification. We report pySM, a framework for false discovery rate (FDR)-controlled metabolite annotation at the level of the molecular sum formula, for high-mass-resolution imaging mass spectrometry ( We introduce a metabolite-signal match score and a target-decoy FDR estimate for spatial metabolomics...
November 14, 2016: Nature Methods
Meghan Zubradt, Paromita Gupta, Sitara Persad, Alan M Lambowitz, Jonathan S Weissman, Silvi Rouskin
Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase...
November 7, 2016: Nature Methods
Alina Selega, Christel Sirocchi, Ira Iosub, Sander Granneman, Guido Sanguinetti
Structure probing coupled with high-throughput sequencing could revolutionize our understanding of the role of RNA structure in regulation of gene expression. Despite recent technological advances, intrinsic noise and high sequence coverage requirements greatly limit the applicability of these techniques. Here we describe a probabilistic modeling pipeline that accounts for biological variability and biases in the data, yielding statistically interpretable scores for the probability of nucleotide modification transcriptome wide...
November 7, 2016: Nature Methods
Elena Rivas, Jody Clements, Sean R Eddy
Many functional RNAs have an evolutionarily conserved secondary structure. Conservation of RNA base pairing induces pairwise covariations in sequence alignments. We developed a computational method, R-scape (RNA Structural Covariation Above Phylogenetic Expectation), that quantitatively tests whether covariation analysis supports the presence of a conserved RNA secondary structure. R-scape analysis finds no statistically significant support for proposed secondary structures of the long noncoding RNAs HOTAIR, SRA, and Xist...
November 7, 2016: Nature Methods
Jing Huang, Sarah Rauscher, Grzegorz Nawrocki, Ting Ran, Michael Feig, Bert L de Groot, Helmut Grubmüller, Alexander D MacKerell
The all-atom additive CHARMM36 protein force field is widely used in molecular modeling and simulations. We present its refinement, CHARMM36m (, with improved accuracy in generating polypeptide backbone conformational ensembles for intrinsically disordered peptides and proteins.
November 7, 2016: Nature Methods
Robert Prevedel, Aart J Verhoef, Alejandro J Pernía-Andrade, Siegfried Weisenburger, Ben S Huang, Tobias Nöbauer, Alma Fernández, Jeroen E Delcour, Peyman Golshani, Andrius Baltuska, Alipasha Vaziri
Although whole-organism calcium imaging in small and semi-transparent animals has been demonstrated, capturing the functional dynamics of large-scale neuronal circuits in awake behaving mammals at high speed and resolution has remained one of the main frontiers in systems neuroscience. Here we present a method based on light sculpting that enables unbiased single- and dual-plane high-speed (up to 160 Hz) calcium imaging as well as in vivo volumetric calcium imaging of a mouse cortical column (0.5 mm × 0.5 mm × 0...
October 31, 2016: Nature Methods
Gaelen T Hess, Laure Frésard, Kyuho Han, Cameron H Lee, Amy Li, Karlene A Cimprich, Stephen B Montgomery, Michael C Bassik
Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously...
October 31, 2016: Nature Methods
Tal Laviv, Benjamin B Kim, Jun Chu, Amy J Lam, Michael Z Lin, Ryohei Yasuda
We describe a red-shifted fluorescence resonance energy transfer (FRET) pair optimized for dual-color fluorescence lifetime imaging (FLIM). This pair utilizes a newly developed FRET donor, monomeric cyan-excitable red fluorescent protein (mCyRFP1), which has a large Stokes shift and a monoexponential fluorescence lifetime decay. When used together with EGFP-based biosensors, the new pair enables simultaneous imaging of the activities of two signaling molecules in single dendritic spines undergoing structural plasticity...
October 31, 2016: Nature Methods
Ibrahim Numanagić, James K Bonfield, Faraz Hach, Jan Voges, Jörn Ostermann, Claudio Alberti, Marco Mattavelli, S Cenk Sahinalp
High-throughput sequencing (HTS) data are commonly stored as raw sequencing reads in FASTQ format or as reads mapped to a reference, in SAM format, both with large memory footprints. Worldwide growth of HTS data has prompted the development of compression methods that aim to significantly reduce HTS data size. Here we report on a benchmarking study of available compression methods on a comprehensive set of HTS data using an automated framework.
October 24, 2016: Nature Methods
Jonathan B Grimm, Brian P English, Heejun Choi, Anand K Muthusamy, Brian P Mehl, Peng Dong, Timothy A Brown, Jennifer Lippincott-Schwartz, Zhe Liu, Timothée Lionnet, Luke D Lavis
Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments...
October 24, 2016: Nature Methods
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