Rxivist logo

Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 92,758 bioRxiv papers from 396,015 authors.

Most downloaded bioRxiv papers, all time

in category biophysics

3,960 results found. For more information, click each entry to expand.

1: Kinetic fingerprints differentiate anti-Aβ therapies
more details view paper

Posted to bioRxiv 22 Oct 2019

Kinetic fingerprints differentiate anti-Aβ therapies
25,654 downloads biophysics

Sara Linse, Tom Scheidt, Katja Bernfur, Michele Vendruscolo, Christopher M. Dobson, Samuel I. A. Cohen, Eimantas Sileikis, Martin Lundquist, Fang Qian, Tiernan O’Malley, Thierry Bussiere, Paul H Weinreb, Catherine K Xu, Georg Meisl, Sean R. A. Devenish, Tuomas P.J. Knowles, Oskar Hansson

The amyloid cascade hypothesis, according to which the self-assembly of amyloid-β peptide (Aβ) is a causative process in Alzheimer’s disease, has driven many therapeutic efforts for the past 20 years. Failures of clinical trials investigating Aβ-targeted therapies have been interpreted as evidence against this hypothesis, irrespective of the characteristics and mechanisms of action of the therapeutic agents, which are highly challenging to assess. We bring together kinetic analysis with quantitative binding measurements to address the mechanisms of action of four clinical stage anti-Aβ antibodies, aducanumab, gantenerumab, bapineuzumab and solanezumab. We reveal and quantify the striking differences of these antibodies on the aggregation kinetics and on the production of oligomeric aggregates, and link these effects to the affinity and stoichiometry of each antibody for monomeric and fibrillar forms of Aβ. Our results uncover that, uniquely amongst these four antibodies, aducanumab dramatically reduces the flux of oligomeric forms of Aβ.

2: Breaking the next Cryo-EM resolution barrier - Atomic resolution determination of proteins!
more details view paper

Posted to bioRxiv 22 May 2020

Breaking the next Cryo-EM resolution barrier - Atomic resolution determination of proteins!
15,687 downloads biophysics

Ka Man Yip, Niels Fischer, Elham Paknia, Ashwin Chari, Holger Stark

Single particle cryo-EM is a powerful method to solve the three-dimensional structures of biological macromolecules. The technological development of electron microscopes, detectors, automated procedures in combination with user friendly image processing software and ever-increasing computational power have made cryo-EM a successful and largely expanding technology over the last decade. At resolutions better than 4 Å , atomic model building starts becoming possible but the direct visualization of true atomic positions in protein structure determination requires significantly higher (< 1.5 Å ) resolution, which so far could not be attained by cryo-EM. The direct visualization of atom positions is essential for understanding protein-catalyzed chemical reaction mechanisms and to study drug- binding and -interference with protein function. Here we report a 1.25 Å resolution structure of apoferritin obtained by cryo-EM with a newly developed electron microscope providing unprecedented structural details. Our apoferritin structure has almost twice the 3D information content of the current world record reconstruction (at 1.54 Å resolution). For the first time in cryo-EM we can visualize individual atoms in a protein, see density for hydrogen atoms and single atom chemical modifications. Beyond the nominal improvement in resolution we can also show a significant improvement in quality of the cryo-EM density map which is highly relevant for using cryo-EM in structure-based drug design. ### Competing Interest Statement The authors have declared no competing interest.

3: Single-particle cryo-EM at atomic resolution
more details view paper

Posted to bioRxiv 22 May 2020

Single-particle cryo-EM at atomic resolution
10,884 downloads biophysics

Takanori Nakane, Abhay Kotecha, Andrija Sente, Greg McMullan, Simonas Masiulis, Patricia M.G.E. Brown, Ioana T. Grigoras, Lina Malinauskaite, Tomas Malinauskas, Jonas Miehling, Lingbo Yu, Dimple Karia, Evgeniya V. Pechnikova, Erwin de Jong, Jeroen Keizer, Maarten Bischoff, Jamie McCormack, Peter Tiemeijer, Steven W Hardwick, Dimitri Y Chirgadze, Garib Murshudov, A. Radu Aricescu, Sjors H. W. Scheres

The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years. However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 Å resolution cryo-EM reconstruction for a prototypical human membrane protein, the β3 GABAA receptor homopentamer. Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 Å resolution reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. Combination of the technological advances described here with further approaches to accelerate data acquisition and improve sample quality provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery. ### Competing Interest Statement A.K., S.M., L.Y., D.K., E.V.P., E.d.J., J.K., M.B., J.M., and P.T are employees of Thermo Fisher Scientific.

4: Transduction of the Geomagnetic Field as Evidenced from Alpha-band Activity in the Human Brain
more details view paper

Posted to bioRxiv 20 Oct 2018

Transduction of the Geomagnetic Field as Evidenced from Alpha-band Activity in the Human Brain
10,069 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.

5: Crystal structure of the 2019-nCoV spike receptor-binding domain bound with the ACE2 receptor
more details view paper

Posted to bioRxiv 20 Feb 2020

Crystal structure of the 2019-nCoV spike receptor-binding domain bound with the ACE2 receptor
9,239 downloads biophysics

Jun Lan, Jiwan Ge, Jinfang Yu, Sisi Shan, Huan Zhou, Shilong Fan, Qi Zhang, Xuanling Shi, Qisheng Wang, Linqi Zhang, Xinquan Wang

A novel and highly pathogenic coronavirus (2019-nCoV) has caused an outbreak in Wuhan city, Hubei province of China since December 2019, and soon spread nationwide and spilled over to other countries around the world. To better understand the initial step of infection at atomic-level, we determined the crystal structure of the 2019-nCoV spike receptor-binding domain (RBD) bound with the cell receptor ACE2 at 2.45 angstrom resolution. The overall ACE2-binding mode of the 2019-nCoV RBD is nearly identical to that of the SARS-CoV RBD, which also utilizes ACE2 as the cell receptor. Structural analysis identified residues in 2019-nCoV RBD critical for ACE2 binding, and majority of which are either highly conserved or shared similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly argue for a convergent evolution between 2019-nCoV and SARS-CoV RBD for improved binding to ACE2 despite of being segregated in different genetic lineages in the betacoronavirus genus. The epitopes of two SARS-CoV antibodies targeting the RBD are also analyzed with the 2019-nCoV RBD, providing insights into future identification of cross-reactive antibodies.

6: Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision
more details view paper

Posted to bioRxiv 05 Dec 2018

Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision
8,608 downloads biophysics

Simao Coelho, Jongho Baek, Matthew S Graus, James M Halstead, Philip R. Nicovich, Kristen Feher, Hetvi Gandhi, Katharina Gaus

Single-molecule localization microscopy (SMLM) promises to provide truly molecular scale images of biological specimens. However, mechanical instabilities in the instrument, readout errors and sample drift constitute significant challenges and severely limit both the useable data acquisition length and the localization accuracy of single molecule emitters. Here, we developed an actively stabilized total internal fluorescence (TIRF) microscope that performs 3D real-time drift corrections and achieves a stability of ≤1 nm. Self-alignment of the emission light path and corrections of readout errors of the camera automate channel alignment and ensure localization precisions of 1-4 nm in DNA origami structures and cells for different labels. We used Feedback SMLM to measure the separation distance of signaling receptors and phosphatases in T cells. Thus, an improved SMLM enables direct distance measurements between molecules in intact cells on the scale between 1-20 nm, potentially replacing Forster resonance energy transfer (FRET) to quantify molecular interactions. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

7: Recording of "sonic attacks" on U.S. diplomats in Cuba spectrally matches the echoing call of a Caribbean cricket
more details view paper

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
8,599 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.

8: RELION-3: new tools for automated high-resolution cryo-EM structure determination
more details view paper

Posted to bioRxiv 19 Sep 2018

RELION-3: new tools for automated high-resolution cryo-EM structure determination
8,599 downloads biophysics

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

9: HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly.
more details view paper

Posted to bioRxiv 21 Feb 2019

HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly.
8,220 downloads biophysics

C. Favard, J. Chojnacki, P. Merida, N. Yandrapalli, J. Mak, C. Eggeling, D. Muriaux

HIV-1 Gag protein self-assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly the phosphatidylinositol (4,5) bisphosphate, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites was determined using super-resolution STED microscopy coupled with scanning Fluorescence Correlation Spectroscopy in living T cells. Analysis of HIV-1 infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data show that Gag is the main driving force to restrict PI(4,5)P2 and cholesterol mobility at the cell plasma membrane. This is first direct evidence showing that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol, as a membrane nano-platform for virus assembly.

10: Real-time cryo-EM data pre-processing with Warp
more details view paper

Posted to bioRxiv 14 Jun 2018

Real-time cryo-EM data pre-processing with Warp
7,138 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.

11: Anisotropic Correction of Beam-induced Motion for Improved Single-particle Electron Cryo-microscopy
more details view paper

Posted to bioRxiv 04 Jul 2016

Anisotropic Correction of Beam-induced Motion for Improved Single-particle Electron Cryo-microscopy
6,762 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.

12: Structure of replicating SARS-CoV-2 polymerase
more details view paper

Posted to bioRxiv 27 Apr 2020

Structure of replicating SARS-CoV-2 polymerase
6,284 downloads biophysics

Hauke S. Hillen, Goran Kokic, Lucas Farnung, Christian Dienemann, Dimitry Tegunov, Patrick Cramer

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. Here we present the cryo-electron microscopic structure of the SARS-CoV-2 RdRp in its replicating form. The structure comprises the viral proteins nsp12, nsp8, and nsp7, and over two turns of RNA template-product duplex. The active site cleft of nsp12 binds the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the RNA duplex as it exits. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged 'sliding poles' that may enable processive replication of the long coronavirus genome. Our results will allow for a detailed analysis of the inhibitory mechanisms used by antivirals such as remdesivir, which is currently in clinical trials for the treatment of coronavirus disease 2019 (COVID-19). ### Competing Interest Statement The authors have declared no competing interest.

13: Achieving Better Than 3 Å Resolution By Single Particle Cryo-EM At 200 keV
more details view paper

Posted to bioRxiv 25 May 2017

Achieving Better Than 3 Å Resolution By Single Particle Cryo-EM At 200 keV
6,045 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.

14: Cryo-EM structure of haemoglobin at 3.2 A determined with the Volta phase plate
more details view paper

Posted to bioRxiv 15 Nov 2016

Cryo-EM structure of haemoglobin at 3.2 A determined with the Volta phase plate
5,032 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.

15: A simple and robust procedure for preparing graphene-oxide cryo-EM grids
more details view paper

Posted to bioRxiv 27 Mar 2018

A simple and robust procedure for preparing graphene-oxide cryo-EM grids
4,814 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.

16: Organization and Regulation of Chromatin by Liquid-Liquid Phase Separation
more details view paper

Posted to bioRxiv 18 Jan 2019

Organization and Regulation of Chromatin by Liquid-Liquid Phase Separation
4,598 downloads biophysics

B.A. Gibson, L.K. Doolittle, L.E. Jensen, N. Gamarra, S. Redding, M.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.

17: Heterochromatin drives organization of conventional and inverted nuclei
more details view paper

Posted to bioRxiv 09 Jan 2018

Heterochromatin drives organization of conventional and inverted nuclei
4,516 downloads biophysics

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

18: Sub-2 Å Ewald Curvature Corrected Single-Particle Cryo-EM
more details view paper

Posted to bioRxiv 21 Apr 2018

Sub-2 Å Ewald Curvature Corrected Single-Particle Cryo-EM
4,440 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.

19: Direct visualization of transcriptional activation by physical enhancer-promoter proximity
more details view paper

Posted to bioRxiv 11 Jan 2017

Direct visualization of transcriptional activation by physical enhancer-promoter proximity
4,405 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.

20: Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.7 Å inside cells
more details view paper

Posted to bioRxiv 05 Jun 2020

Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.7 Å inside cells
4,262 downloads biophysics

Dimitry Tegunov, Liang Xue, Christian Dienemann, Patrick Cramer, Julia Mahamid

Cryo-electron microscopy (cryo-EM) enables macromolecular structure determination in vitro and in situ . In addition to aligning individual particles, accurate registration of sample motion and 3D deformation during exposures is crucial for achieving high resolution. Here we describe M , a software tool that establishes a reference-based, multi-particle refinement framework for cryo-EM data and improves the results of structure determination. M provides a unified optimization framework for both in vitro frame series and in situ tomographic tilt series data. We show that tilt series data can provide the same resolution as frame series, indicating that the alignment step no longer limits the resolution obtainable from tomographic data. In combination with Warp and RELION, M improves upon previous methods, and resolves a 70S ribosome bound to an antibiotic inside bacterial cells at a nominal resolution of 3.7 Å. Thus, computational tools are now available to resolve structures from tomographic in situ cryo-EM data at residue level. ### Competing Interest Statement The authors have declared no competing interest.

Previous page 1 2 3 4 5 . . . 198 Next page

PanLingua

Sign up for the Rxivist weekly newsletter! (Click here for more details.)


News