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Results 1 through 20 out of 2480

in category biophysics


1: Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

Stephen Graf Brohawn, David M Kern et al.

1,491 downloads (posted 14 Oct 2019)

Cation-chloride-cotransporters (CCCs) catalyze transport of Cl- with K+ and/or Na+ across cellular membranes. CCCs play roles in volume regulation, neural development and function, audition, blood pressure regulation, and renal function. CCCs are targets of drugs including loop diuretics and their disruption is implicated in pathophysiologies including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the cryo-EM structure of a CCC, the Mus musculus K+-Cl- cotransporter KCC4, in lipid nanodiscs. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We further identify substrate K+ and Cl- binding sites and provide a structural explanation for differences in substrate specificity and transport ratio between CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family.


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

Cyril Favard, Jakub Chojnacki et al.

993 downloads (posted 21 Feb 2019)

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 S...


3: The cryo-EM structure of human CST reveals a two-megadalton decameric assembly bound to telomeric DNA

Ci Ji Lim, Alexandra T. Barbour et al.

906 downloads (posted 23 Oct 2019)

The single-stranded DNA-binding CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and genome-wide replication recovery, processes that are critical for genome stability. Here, we report the 2.95 Å cryo-EM structure of human CST bound to telomeric single-stranded DNA, which unexpectedly assembles as a decameric supercomplex. The atomic model of the 134 kDa CTC1, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. In situ arrangements of STN1 and TEN1 ar...


4: Comment on 'Response to comment on 'Valid molecular dynamics simulations of human hemoglobin require a surprisingly large box size''

Vytautas Gapsys, Bert L. de Groot

753 downloads (posted 25 Oct 2019)

We recently expressed three major concerns about a 2018 article of El Hage et al. about a claimed effect of the box size in molecular dynamics simulations of hemoglobin. In the response of the authors to our comment, none of these concerns have been addressed, yet the authors maintain their original conclusions. Here, we challenge those conclusions and provide additional data that reestablish our original concerns. In addition, we identified six additional flaws in the response from El Hage et al. as well as a number of...


5: Composition dependent phase separation underlies directional flux through the nucleolus

Joshua A Riback, Lian Zhu et al.

701 downloads (posted 22 Oct 2019)

Intracellular bodies such as nucleoli, Cajal bodies, and various signaling assemblies, represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions, particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions (IDRs), are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal su...


6: A complex structure of arrestin-2 bound to a G protein-coupled receptor

Wanchao Yin, Zhihai Li et al.

668 downloads (posted 29 Oct 2019)

Arrestins comprise a family of signal regulators of G-protein-coupled receptors (GPCRs), which include arrestins 1 to 4. While arrestins 1 and 4 are visual arrestins dedicated to rhodopsin, arrestins 2 and 3 (Arr2 and Arr3) are β-arrestins known to regulate many nonvisual GPCRs. The dynamic and promiscuous coupling of Arr2 to nonvisual GPCRs has posed technical challenges to tackle the basis of arrestin binding to GPCRs. Here we report the structure of Arr2 in complex with neurotensin receptor 1 (NTSR1), which reveals a...


7: The SAMPL6 SAMPLing challenge: Assessing the reliability and efficiency of binding free energy calculations

Andrea Rizzi, Travis Jensen et al.

636 downloads (posted 07 Oct 2019)

Approaches for computing small molecule binding free energies based on molecular simulations are now regularly being employed by academic and industry practitioners to study receptor-ligand systems and prioritize the synthesis of small molecules for ligand design. Given the variety of methods and implementations available, it is natural to ask how the convergence rates and final predictions of these methods compare. In this study, we describe the concept and results for the SAMPL6 SAMPLing challenge, the first challenge...


8: Micro-scale fluid behavior during cryo-EM sample blotting

Maxim Armstrong, Bong-Gyoon Han et al.

592 downloads (posted 02 Oct 2019)

Blotting has been the standard technique for preparing aqueous samples for single-particle electron cryo-microscopy (cryo-EM) for over three decades. This technique removes excess solution from a TEM grid by pressing absorbent filter paper against the specimen prior to vitrification. However, this standard technique produces vitreous ice with inconsistent thickness from specimen to specimen and from region to region within the same specimen, the reasons for which are not understood. Here, high-speed interference-contras...


9: A cost-efficient open source laser engine for microscopy

Daniel Schroeder, Joran Deschamps et al.

591 downloads (posted 15 Oct 2019)

Scientific-grade lasers are costly components of modern microscopes. For high-power applications, such as single-molecule localization microscopy, their price can become prohibitive. Here, we present an open-source high-power laser engine that can be built for a fraction of the cost. It uses affordable, yet powerful laser diodes at wavelengths of 405 nm, 488 nm and 640 nm and optionally a 561 nm diode-pumped solid-state laser. The light is delivered to the microscope via an agitated multimode fiber in order to suppress ...


10: Chromosome organization by one-sided and two-sided loop extrusion

Edward J Banigan, Aafke van den Berg et al.

571 downloads (posted 22 Oct 2019)

SMC complexes organize chromatin throughout the cell cycle across many cell types. Experiments indicate that this is achieved by an energy-consuming process known as loop extrusion, in which SMC complexes, such as condensin or cohesin, reel in DNA/chromatin, extruding and progressively growing a DNA/chromatin loop. Theoretical modeling assuming two-sided loop extrusion has successfully reproduced key features of chromatin organization across different organisms. Recent in vitro single-molecule experiments confirmed that...


11: High Yield Monolayer Graphene Grids for Near-Atomic Resolution Cryo-Electron Microscopy

Yimo Han, Xiao Fan et al.

567 downloads (posted 01 Nov 2019)

Cryogenic electron microscopy (cryo-EM) has become one of the most powerful techniques to reveal the atomic structures and working mechanisms of biological macromolecules. New designs of the cryo-EM grids, aimed at preserving thin, uniform vitrified ice and improving protein adsorption, have been considered a promising approach to achieving higher resolution with the minimal amount of materials and data. Here, we describe a method for preparing graphene cryo-EM grids with 99% monolayer graphene coverage that allows for ...


12: Amino and PEG-Amino Graphene Oxide Grids Enrich and Protect Samples for High-resolution Single Particle Cryo-electron Microscopy

Feng Wang, Zanlin Yu et al.

533 downloads (posted 22 Oct 2019)

Cryo-EM samples prepared using the traditional methods often suffer from too few particles, poor particle distribution, or strongly biased orientation, or damage from the air-water interface. Here we report that functionalization of graphene oxide (GO) coated grids with amino groups concentrates samples on the grid with improved distribution and orientation. By introducing a PEG spacer, particles are kept away from both the GO surface and the air-water interface, protecting them from potential denaturation.


13: DMD-based super-resolution structured illumination microscopy visualizes live cell dynamics at high speed and low cost

Alice Sandmeyer, Mario Lachetta et al.

499 downloads (posted 08 Oct 2019)

Structured illumination microscopy (SIM) is among the most widely used super-resolution fluorescence microscopy techniques for visualizing the dynamics of cellular organelles, such as mitochondria, the endoplasmic reticulum, or the cytoskeleton. In its most wide-spread implementation, SIM relies on the creation of an interference pattern at the diffraction limit using the coherent addition of laser beams created by a diffraction pattern. Spatial light modulators based on liquid crystal displays allow SIM microscopes to ...


14: Membrane constriction and thinning by sequential ESCRT-III polymerization

Henry C Nguyen, Nathaniel Talledge et al.

488 downloads (posted 11 Oct 2019)

The Endosomal Sorting Complexes Required for Transport (ESCRTs) mediate diverse membrane remodeling events. These activities typically require ESCRT-III proteins to stabilize negatively-curved membranes, although recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane curvature shaping by these proteins remains poorly understood. To learn how E...


15: RELION-3: new tools for automated high-resolution cryo-EM structure determination

Jasenko Zivanov, Takanori Nakane et al.

477 downloads (posted 19 Sep 2018)

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 hig...


16: Amyloid structure determination in RELION-3.1

Sjors Scheres

438 downloads (posted 29 Oct 2019)

Helical reconstruction in RELION is increasingly used to determine atomic structures of amyloid filaments from electron cryo-microscopy (cryo-EM) images. However, because the energy landscape of amyloid refinements is typically fraught with local optima, amyloid structure determination is often difficult. This paper aims to help RELION users in this process. It discusses aspects of helical reconstruction that are specific to amyloids; it illustrates the problem of local optima in refinement and how to detect these; and ...


17: MINFLUX nanoscopy delivers multicolor nanometer 3D-resolution in (living) cells

Klaus C. Gwosch, Jasmin K. Pape et al.

436 downloads (posted 13 Aug 2019)

The ultimate goal of biological superresolution fluorescence microscopy is to provide three-dimensional resolution at the size scale of a fluorescent marker. Here, we show that, by localizing individual switchable fluorophores with a probing doughnut-shaped excitation beam, MINFLUX nanoscopy provides 1 to 3 nanometer resolution in fixed and living cells. This progress has been facilitated by approaching each fluorophore iteratively with the probing doughnut minimum, making the resolution essentially uniform and isotropi...


18: Cell surface mechanics gate stem cell differentiation

Martin Bergert, Sergio Lembo et al.

425 downloads (posted 09 Oct 2019)

Cell differentiation typically occurs with concomitant shape transitions to enable specialized functions. To adopt a different shape, cells need to change the mechanical properties of their surface. However, whether conversely cell surface mechanics control the process of differentiation has been relatively unexplored. Here, we show that membrane mechanics gate the exit from naïve pluripotency of mouse embryonic stem cells. By measuring membrane tension during differentiation, we find that naïve stem cells release their...


19: How to define and optimize axial resolution in light-sheet microscopy: a simulation-based approach

Elena Remacha, Lars Friedrich et al.

422 downloads (posted 27 Sep 2019)

'How thick is your light sheet?' is a question that has been asked frequently after talks showing impressive renderings of 3D data acquired by a light-sheet microscope. This question is motivated by the fact that most of the time the thickness of the light-sheet is uniquely associated to the axial resolution of the microscope. However, the link between lightsheet thickness and axial resolution has never been systematically assessed and it is still unclear how both are connected. The question is not trivial because commo...


20: Mechanisms of activation and desensitization of full-length glycine receptor in membranes

Arvind kumar, Sandip Basak et al.

421 downloads (posted 30 Sep 2019)

Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyR) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structural studies have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unknown. Here, we present Cryo-EM structures of the full-length GlyR protein reconstituted into lipid nanodiscs that are cap...