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Most downloaded bioRxiv papers, all time

in category biophysics

2,039 results found. For more information, click each entry to expand.

1: Transduction of the Geomagnetic Field as Evidenced from Alpha-band Activity in the Human Brain
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Posted to bioRxiv 20 Oct 2018

Transduction of the Geomagnetic Field as Evidenced from Alpha-band Activity in the Human Brain
7,683 downloads biophysics

Connie X Wang, Isaac A Hilburn, Daw-An Wu, Yuki Mizuhara, Christopher P Cousté, Jacob N. H. Abrahams, Sam E Bernstein, Ayumu Matani, Shinsuke Shimojo, Joseph L Kirschvink

Magnetoreception, the perception of the geomagnetic field, is a sensory modality well-established across all major groups of vertebrates and some invertebrates, but its presence in humans has been tested rarely, yielding inconclusive results. We report here a strong, specific human brain response to ecologically-relevant rotations of Earth-strength magnetic fields. Following geomagnetic stimulation, a drop in amplitude of EEG alpha oscillations (8-13 Hz) occurred in a repeatable manner. Termed alpha event-related desynchronization (alpha-ERD), such a response is associated with sensory and cognitive processing of external stimuli. Biophysical tests showed that the neural response was sensitive to the dynamic components and axial alignment of the field but also to the static components and polarity of the field. This pattern of results implicates ferromagnetism as the biophysical basis for the sensory transduction and provides a basis to start the behavioral exploration of human magnetoreception.

2: Recording of "sonic attacks" on U.S. diplomats in Cuba spectrally matches the echoing call of a Caribbean cricket
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Posted to bioRxiv 04 Jan 2019

Recording of "sonic attacks" on U.S. diplomats in Cuba spectrally matches the echoing call of a Caribbean cricket
7,683 downloads biophysics

Alexander L Stubbs, Fernando Montealegre-Z

Beginning in late 2016, diplomats posted to the United States embassy in Cuba began to experience unexplained health problems including ear pain, tinnitus, vertigo, and cognitive difficulties which reportedly began after they heard strange noises in their homes or hotel rooms. In response, the U.S. government dramatically reduced the number of diplomats posted at the U.S. embassy in Havana. U.S. officials initially believed a sonic attack might be responsible for their ailments. The sound linked to these attacks, which has been described as a high-pitched beam of sound, was recorded by U.S. personnel in Cuba and released by the Associated Press (AP). Because these recordings are the only available non-medical evidence of the sonic attacks, much attention has focused on identifying health problems and the origin of the acoustic signal. As shown here, the calling song of the Indies short-tailed cricket (Anurogryllus celerinictus) matches, in nuanced detail, the AP recording in duration, pulse repetition rate, power spectrum, pulse rate stability, and oscillations per pulse. The AP recording also exhibits frequency decay in individual pulses, a distinct acoustic signature of cricket sound production. While the temporal pulse structure in the recording is unlike any natural insect source, when the cricket call is played on a loudspeaker and recorded indoors, the interaction of reflected sound pulses yields a sound virtually indistinguishable from the AP sample. This provides strong evidence that an echoing cricket call, rather than a sonic attack or other technological device, is responsible for the sound in the released recording. Although the causes of the health problems reported by embassy personnel are beyond the scope of this paper, our findings highlight the need for more rigorous research into the source of these ailments, including the potential psychogenic effects, as well as possible physiological explanations unrelated to sonic attacks.

3: Anisotropic Correction of Beam-induced Motion for Improved Single-particle Electron Cryo-microscopy
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Posted to bioRxiv 04 Jul 2016

Anisotropic Correction of Beam-induced Motion for Improved Single-particle Electron Cryo-microscopy
5,780 downloads biophysics

Shawn Q. Zheng, Eugene Palovcak, Jean-Paul Armache, Yifan Cheng, David A Agard

Correction of electron beam-induced sample motion is one of the major factors contributing to the recent resolution breakthroughs in cryo-electron microscopy. Improving the accuracy and efficiency of motion correction can lead to further resolution improvement. Based on observations that the electron beam induces doming of the thin vitreous ice layer, we developed an algorithm to correct anisotropic image motion at the single pixel level across the whole frame, suitable for both single particle and tomographic images. Iterative, patch-based motion detection is combined with spatial and temporal constraints and dose weighting. The multi-GPU accelerated program, MotionCor2, is sufficiently fast to keep up with automated data collection. The result is an exceptionally robust strategy that can work on a wide range of data sets, including those very close to focus or with very short integration times, obviating the need for particle polishing. Application significantly improves Thon ring quality and 3D reconstruction resolution.

4: Achieving Better Than 3 Å Resolution By Single Particle Cryo-EM At 200 keV
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Posted to bioRxiv 25 May 2017

Achieving Better Than 3 Å Resolution By Single Particle Cryo-EM At 200 keV
5,284 downloads biophysics

Mark A Herzik, Mengyu Wu, Gabriel C Lander

Technical and methodological advances in single-particle cryo-electron microscopy (cryo-EM) have expanded the technique into a resolution regime that was previously only attainable by X-ray crystallography. Although single-particle cryo-EM has proven to be a useful technique for determining the structures of biomedically relevant molecules at near-atomic resolution, nearly 98% of the structures resolved to better than 4 Å resolution have been determined using 300 keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain cryo-EM reconstructions of macromolecular complexes at a range of sizes to better than 3 Å resolution using a 200 keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules, features previously thought only to be resolvable using TEMs operating at 300 keV.

5: Cryo-EM structure of haemoglobin at 3.2 A determined with the Volta phase plate
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Posted to bioRxiv 15 Nov 2016

Cryo-EM structure of haemoglobin at 3.2 A determined with the Volta phase plate
4,851 downloads biophysics

Maryam Khoshouei, Mazdak Radjainia, Wolfgang Baumeister, Radostin Danev

With the advent of direct electron detectors, the perspectives of cryo-electron microscopy (cryo-EM) have changed in a profound way (ref. 1). These cameras are superior to previous detectors in coping with the intrinsically low contrast of radiation-sensitive organic materials embedded in amorphous ice, and so they have enabled the structure determination of several macromolecular assemblies to atomic or near-atomic resolution. According to one theoretical estimation, a few thousand images should suffice for calculating the structure of proteins as small as 17 kDa at 3 A resolution (ref. 2). In practice, however, we are still far away from this theoretical ideal. Thus far, protein complexes that have been successfully reconstructed to high-resolution by single particle analysis (SPA) have molecular weights of ~100 kDa or larger (ref. 3). Here, we report the use of Volta phase plate in determining the structure of human haemoglobin (64 kDa) at 3.2 A. Our results demonstrate that this method can be applied to complexes that are significantly smaller than those previously studied by conventional defocus-based approaches. Cryo-EM is now close to becoming a fast and cost-effective alternative to crystallography for high-resolution protein structure determination.

6: RELION-3: new tools for automated high-resolution cryo-EM structure determination
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Posted to bioRxiv 19 Sep 2018

RELION-3: new tools for automated high-resolution cryo-EM structure determination
4,744 downloads biophysics

Jasenko Zivanov, Takanori Nakane, Bjorn Forsberg, Dari Kimanius, Wim J.H. Hagen, Erik Lindahl, Sjors Scheres

Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher-resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 Å compared to previous RELION versions.

7: Real-time cryo-EM data pre-processing with Warp
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Posted to bioRxiv 14 Jun 2018

Real-time cryo-EM data pre-processing with Warp
4,100 downloads biophysics

Dimitry Tegunov, Patrick Cramer

The acquisition of cryo-electron microscopy (cryo-EM) data from biological specimens is currently largely uncoupled from subsequent data evaluation, correction and processing. Therefore, the acquisition strategy is difficult to optimize during data collection, often leading to suboptimal microscope usage and disappointing results. Here we provide Warp, a software for real-time evaluation, correction, and processing of cryo-EM data during their acquisition. Warp evaluates and monitors key parameters for each recorded micrograph or tomographic tilt series in real time. Warp also rapidly corrects micrographs for global and local motion, and estimates the local defocus with the use of novel algorithms. The software further includes a deep learning-based particle picking algorithm that rivals human accuracy to make the pre-processing pipeline truly automated. The output from Warp can be directly fed into established tools for particle classification and 3D image reconstruction. In a benchmarking study we show that Warp automatically processed a published cryo-EM data set for influenza virus hemagglutinin, leading to an improvement of the nominal resolution from 3.9 Å to 3.2 Å. Warp is easy to install, computationally inexpensive, and has an intuitive and streamlined user interface.

8: Direct visualization of transcriptional activation by physical enhancer-promoter proximity
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Posted to bioRxiv 11 Jan 2017

Direct visualization of transcriptional activation by physical enhancer-promoter proximity
4,079 downloads biophysics

Hongtao Chen, Miki Fujioka, James B. Jaynes, Thomas Gregor

A long-standing question in metazoan gene regulation is how remote enhancers communicate with their target promoters over long distances. Combining genome editing and quantitative live imaging we simultaneously visualize physical enhancer-promoter communication and transcription in Drosophila embryos. Enhancers regulating pair rule stripes of even-skipped expression activate transcription of a reporter gene over a distance of 150 kb. We show in individual cells that activation only occurs after the enhancer comes into close proximity with its regulatory target and that upon dissociation transcription ceases almost immediately. We further observe distinct topological conformations of the eve locus, depending on the spatial identity of the activating stripe enhancer. In addition, long-range activation results in transcriptional competition at the endogenous eve locus, causing corresponding developmental defects. Overall, we demonstrate that sustained physical proximity and enhancer-promoter engagement are required for enhancer action, and we provide a path to probe the implications of long-range regulation on cellular fates.

9: Sub-2 Å Ewald Curvature Corrected Single-Particle Cryo-EM
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Posted to bioRxiv 21 Apr 2018

Sub-2 Å Ewald Curvature Corrected Single-Particle Cryo-EM
4,054 downloads biophysics

Yong Zi Tan, Sriram Aiyer, Mario Mietzsch, Joshua A. Hull, Robert McKenna, Joshua Grieger, R. Jude Samulski, Timothy S. Baker, Mavis Agbandje-McKenna, Dmitry Lyumkis

Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we have exploited several technical solutions to improve resolution, including sub-Angstrom pixelation, per-particle CTF refinement, and most notably a correction for Ewald sphere curvature. The application of these methods on micrographs recorded on a base model Titan Krios enabled structure determination at ~1.86-Å resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle.

10: Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2
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Posted to bioRxiv 19 Jun 2016

Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2
3,908 downloads biophysics

Dari Kimanius, Björn O Forsberg, Sjors H.W. Scheres, Erik Lindahl

By reaching near-atomic resolution for a wide range of specimens, single-particle cryo-EM structure determination is transforming struc- tural biology. However, the necessary calculations come at increased computational costs, introducing a bottleneck that is currently limiting throughput and the development of new methods. Here, we present an implementation of the relion image processing software that uses graphics processors (GPUs) to address the most computationally intensive steps of its cryo-EM structure determination workflow. Both image classification and high-resolution refinement have been accelerated up to 40-fold, and template-based particle selection has been accelerated almost 1000-fold on desktop hardware. Memory requirements on GPUs have been reduced to fit widely available hard- ware, and we show that the use of single precision arithmetic does not adversely affect results. This enables high-resolution cryo-EM struc- ture determination in a matter of days on a single workstation.

11: Heterochromatin drives organization of conventional and inverted nuclei
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Posted to bioRxiv 09 Jan 2018

Heterochromatin drives organization of conventional and inverted nuclei
3,610 downloads biophysics

Martin Falk, Yana Feodorova, Natasha Naumova, Maxim Imakaev, Bryan R. Lajoie, Heinrich Leonhardt, Boris Joffe, Job Dekker, Geoffrey Fudenberg, Irina Solovei, Leonid A. Mirny

The mammalian cell nucleus displays a remarkable spatial segregation of active euchromatic from inactive heterochromatic genomic regions. In conventional nuclei, euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery. In contrast, rod photoreceptors in nocturnal mammals have inverted nuclei, with a dense heterochromatic core and a thin euchromatic outer shell. This inverted architecture likely converts rod nuclei into microlenses to facilitate nocturnal vision, and may relate to the absence of particular proteins that tether heterochromatin to the lamina. However, both the mechanism of inversion and the role of interactions between different types of chromatin and the lamina in nuclear organization remain unknown. To elucidate this mechanism we performed Hi-C and microscopy on cells with inverted nuclei and their conventional counterparts. Strikingly, despite the inversion evident in microscopy, both types of nuclei display similar Hi-C maps. To resolve this paradox we developed a polymer model of chromosomes and found a universal mechanism that reconciles Hi-C and microscopy for both inverted and conventional nuclei. Based solely on attraction between heterochromatic regions, this mechanism is sufficient to drive phase separation of euchromatin and heterochromatin and faithfully reproduces the 3D organization of inverted nuclei. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. To further test our models, we eliminated lamina interactions in models of conventional nuclei and found that this triggers a spontaneous process of inversion that qualitatively reproduces the pathway of morphological changes during nuclear inversion in vivo. Together, our experiments and modeling suggest that interactions among heterochromatic regions are central to phase separation of the active and inactive genome in inverted and conventional nuclei, while interactions with the lamina are essential for building the conventional architecture from these segregated phases. Ultimately our data suggest that an inverted organization constitutes the default state of nuclear architecture.

12: A simple and robust procedure for preparing graphene-oxide cryo-EM grids
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Posted to bioRxiv 27 Mar 2018

A simple and robust procedure for preparing graphene-oxide cryo-EM grids
3,417 downloads biophysics

Eugene Palovcak, Feng Wang, Shawn Q. Zheng, Zanlin Yu, Sam Li, David Bulkley, David A Agard, Yifan Cheng

Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited its widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids are suitable for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and providing partial protection from the air-water interface. Additionally, the signal from images of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.

13: CTFFIND4: Fast and accurate defocus estimation from electron micrographs
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Posted to bioRxiv 16 Jun 2015

CTFFIND4: Fast and accurate defocus estimation from electron micrographs
3,227 downloads biophysics

Alexis Rohou, Nikolaus Grigorieff

CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Defocus parameters are estimated by fitting a model of the microscope?s contrast transfer function (CTF) to an image?s amplitude spectrum. Here we describe modifications to the algorithm which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates. We show that this new version preserves the accuracy of the original algorithm while allowing for higher throughput. We also describe a measure of the quality of the fit as a function of spatial frequency and suggest this can be used to define the highest resolution at which CTF oscillations were successfully modeled.

14: NanoJ-SQUIRREL: quantitative mapping and minimisation of super-resolution optical imaging artefacts
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Posted to bioRxiv 02 Jul 2017

NanoJ-SQUIRREL: quantitative mapping and minimisation of super-resolution optical imaging artefacts
3,152 downloads biophysics

Siân Culley, David Albrecht, Caron Jacobs, Pedro Matos Pereira, Christophe Leterrier, Jason Mercer, Ricardo Henriques

Most super-resolution microscopy methods depend on steps that contribute to the formation of image artefacts. Here we present NanoJ-SQUIRREL, an ImageJ-based analytical approach providing a quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same focal volume, this approach generates a quantitative map of super-resolution defects, as well as methods for their correction. To illustrate its broad applicability to super-resolution approaches we apply our method to Localization Microscopy, STED and SIM images of a variety of in-cell structures including microtubules, poxviruses, neuronal actin rings and clathrin coated pits. We particularly focus on single-molecule localisation microscopy, where super-resolution reconstructions often feature imperfections not present in the original data. By showing the quantitative evolution of data quality over these varied sample preparation, acquisition and super-resolution methods we display the potential of NanoJ-SQUIRREL to guide optimization of super-resolution imaging parameters.

15: The deadly touch: protein denaturation at the water-air interface and how to prevent it
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Posted to bioRxiv 25 Aug 2018

The deadly touch: protein denaturation at the water-air interface and how to prevent it
2,906 downloads biophysics

Edoardo D'Imprima, Davide Floris, Mirko Joppe, Ricardo Sánchez, Martin Grininger, Werner Kühlbrandt

Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation of the protein. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions are those facing the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.

16: Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION
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Posted to bioRxiv 22 Mar 2018

Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION
2,849 downloads biophysics

Takanori Nakane, Dari Kimanius, Erik Lindahl, Sjors H.W. Scheres

Macromolecular complexes that exhibit continuous forms of structural flexibility pose a challenge for many existing tools in cryo-EM single-particle analysis. We describe a new tool, called multi-body refinement, which models flexible complexes as a user-defined number of rigid bodies that move independently from each other. Using separate focused refinements with iteratively improved partial signal subtraction, the new tool generates improved reconstructions for each of the defined bodies in a fully automated manner. Moreover, using principal component analysis on the relative orientations of the bodies over all particles in the data set, we generate movies that describe the most important motions in the data. Our results on two test cases, a cytoplasmic ribosome from Plasmodium falciparum, and the spliceosomal B-complex from yeast, illustrate how multi-body refinement can be useful to gain unique insights into the structure and dynamics of large and flexible macromolecular complexes.

17: Gctf: real-time CTF determination and correction
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Posted to bioRxiv 12 Jul 2015

Gctf: real-time CTF determination and correction
2,757 downloads biophysics

Kai Zhang

Accurate estimation of the contrast transfer function (CTF) is critical for a near-atomic resolution cryo electron microscopy (cryoEM) reconstruction. Here, I present a GPU-accelerated computer program, Gctf, for accurate and robust, real-time CTF determination. Similar to alternative programs, the main target of Gctf is to maximize the cross-correlation of a simulated CTF with the power spectra of observed micrographs after background reduction. However, novel approaches in Gctf improve both speed and accuracy. In addition to GPU acceleration, a fast ?1-dimensional search plus 2-dimensional refinement (1S2R)? procedure significantly speeds up Gctf. Based on the global CTF determination, the local defocus for each particle and for single frames of movies is accurately refined, which improves CTF parameters of all particles for subsequent image processing. Novel diagnosis method using equiphase averaging(EFA) and self-consistency verification procedures have also been implemented in the program for practical use, especially for aims of near-atomic reconstruction. Gctf is an independent program and the outputs can be easily imported into other cryoEM software such as Relion and Frealign. The results from several representative datasets are shown and discussed in this paper.

18: Organization and Regulation of Chromatin by Liquid-Liquid Phase Separation
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Posted to bioRxiv 18 Jan 2019

Organization and Regulation of Chromatin by Liquid-Liquid Phase Separation
2,646 downloads biophysics

Bryan A Gibson, Lynda K Doolittle, Liv E Jensen, Nathan Gamarra, Sy Redding, Michael K. Rosen

Genomic DNA is highly compacted in the nucleus of eukaryotic cells as a nucleoprotein assembly called chromatin. The basic unit of chromatin is the nucleosome, where ~146 base pair increments of the genome are wrapped and compacted around the core histone proteins. Further genomic organization and compaction occur through higher order assembly of nucleosomes. This organization regulates many nuclear processes, and is controlled in part by histone post-transtranslational modifications and chromatin-binding proteins. Mechanisms that regulate the assembly and compaction of the genome remain unclear. Here we show that in the presence of physiologic concentrations of mono- and divalent salts, histone tail-driven interactions drive liquid-liquid phase separation (LLPS) of nucleosome arrays, resulting in substantial condensation. Phase separation of nucleosomal arrays is inhibited by histone acetylation, whereas histone H1 promotes phase separation, further compaction, and decreased dynamics within droplets, mirroring the relationship between these modulators and the accessibility of the genome in cells. These results indicate that under physiologically relevant conditions, LLPS is an intrinsic behavior of the chromatin polymer, and suggest a model in which the condensed phase reflects a genomic 'ground state' that can produce chromatin organization and compaction in vivo. The dynamic nature of this state could enable known modulators of chromatin structure, such as post-translational modifications and chromatin binding proteins, to act upon it and consequently control nuclear processes such as transcription and DNA repair. Our data suggest an important role for LLPS of chromatin in the organization of the eukaryotic genome.

19: The Rosetta all-atom energy function for macromolecular modeling and design
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Posted to bioRxiv 07 Feb 2017

The Rosetta all-atom energy function for macromolecular modeling and design
2,585 downloads biophysics

Rebecca F. Alford, Andrew Leaver-Fay, Jeliazko R Jeliazkov, Matthew J O'Meara, Frank P DiMaio, Hahnbeom Park, Maxim V Shapovalov, P. Douglas Renfrew, Vikram K Mulligan, Kalli Kappel, Jason W Labonte, Michael S Pacella, Richard Bonneau, Philip Bradley, Roland L. Dunbrack, Rhiju Das, David Baker, Brian Kuhlman, Tanja Kortemme, Jeffrey J Gray

Over the past decade, the Rosetta biomolecular modeling suite has informed diverse biological questions and engineering challenges ranging from interpretation of low-resolution structural data to design of nanomaterials, protein therapeutics, and vaccines. Central to Rosetta's success is the energy function: a model parameterized from small molecule and X-ray crystal structure data used to approximate the energy associated with each biomolecule conformation. This paper describes the mathematical models and physical concepts that underlie the latest Rosetta energy function, Aasgard2017. Applying these concepts, we explain how to use Rosetta energies to identify and analyze the features of biomolecular models. Finally, we discuss the latest advances in the energy function that extend capabilities from soluble proteins to also include membrane proteins, peptides containing non-canonical amino acids, carbohydrates, nucleic acids, and other macromolecules.

20: Microfluidic protein isolation and sample preparation for high resolution cryo-EM
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Posted to bioRxiv 21 Feb 2019

Microfluidic protein isolation and sample preparation for high resolution cryo-EM
2,550 downloads biophysics

Claudio Schmidli, Stefan Albiez, Luca Rima, Ricardo Righetto, Inayatulla Mohammed, Paola Oliva, Lubomir Kovacik, Henning Stahlberg, Thomas Braun

High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousands to a few million protein particles required for structure-determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than one microliter of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens new opportunities for the isolation of sensitive, endogenous protein complexes.

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