Biology subjects

Beckers, M.

Publications and source records attributed to Beckers, M..

4 recordsLinked to original sources

bioRxiv2021

Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution

Electron cryo-microscopy (cryo-EM) is becoming one of the routine tools for structure determination of biological macromolecules. Commonly, molecular images are obtained by conventional transmission electron microcopy (CTEM) using underfocus and subsequently computationally combined into a high-resolution 3D structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC-STEM to the cryo-EM test specimen of tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination at 3.5 [A] resolution using single-particle based helical reconstruction. A series of cryo-EM TMV maps was resolved at near-atomic resolution taken at different convergence semi-angle (CSA) beams and share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. The associated map B-factors from iDPC-STEM match those obtained by CTEM recordings using 2nd generation direct electron detection devices. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.

biophysics↗

bioRxiv2020

Adaptive FDR thresholding of Fourier shell correlation for resolution estimation of cryo-EM maps

Fourier shell correlation (FSC) has become a standard quantity for resolution estimation in electron cryo-microscopy. However, the resolution determination step is still subjective and not fully automated as it involves a series of map interventions before FSC computation and includes the selection of a common threshold. Here, we apply the statistical methods of permutation sampling and false discovery rate (FDR) control to the resolution-dependent correlation measure. The approach allows fully automated and mask-free resolution determination based on adaptive thresholding of FSC curves. We demonstrate the applicability for global, local and directional resolution estimation and show that the developed criterion termed FDR-FSC gives realistic resolution estimates based on a statistical significance criterion while eliminating the need of any map manipulations. The algorithms are implemented in a user-friendly GUI based software tool termed SPoC (https://github.com/MaximilianBeckers/SPOC).

biophysics↗

bioRxiv2019

Elucidation of the viral disassembly switch of tobacco mosaic virus

Stable capsid structures of viruses protect viral RNA while they also require controlled disassembly for releasing the viral genome in the host cell. A detailed understanding of viral disassembly processes and the involved structural switches is still lacking. Biochemically, this process has been extensively studied using the tobacco mosaic virus model system and carboxylate interactions have been proposed to play a critical part in this process. Here, we present two cryo-EM structures of the helical TMV assembly at 2.1 and 2.0 [A] resolution in conditions of high Ca2+ concentration at low pH and in water. Based on our atomic models, we identified the conformational details of the disassembly switch mechanism: in high Ca2+/acidic pH environment the virion is stabilized between neighboring subunits through carboxyl groups E95 and E97 in close proximity to a Ca2+ binding site. Upon increase in pH and lower Ca2+ levels, mutual repulsion of the E95/E97 pair and Ca2+ removal destabilize the network of interactions at lower radius and release the switch of virus disassembly. Our TMV structures revealed the conformational details for one of the reference systems of viral assembly/disassembly and provide the mechanistic explanation of a plethora of experimental results that were acquired over decades.\n\nSignificance StatementTobacco mosaic virus presents the text-book example of virus structure and RNA release from a viral capsid through disassembly. Despite the wealth of structural and biochemical data on the assembly/disassembly properties generated from more than 80 years of research, the atomic-resolution structural details of the proposed conformational changes have not been resolved to date. The here determined high-resolution cryo-EM structures reveal the conformational details of the molecular disassembly switch. When the virus enters the cell, carboxylate repulsion and loss of calcium-ion coordination destabilize the switch region and can trigger RNA release through virus disassembly. The two determined structural states resolve a long-standing question on environment-driven virus disassembly switches.

molecular biology↗

bioRxiv2018

Thresholding of cryo-EM density maps by false discovery rate control

Cryo-EM now commonly generates close-to-atomic resolution as well as intermediate resolution maps from macromolecules observed in isolation and in situ. Interpreting these maps remains a challenging task due to poor signal in the highest resolution shells and the necessity to select a threshold for density analysis. In order to facilitate this process, we developed a statistical framework for the generation of confidence maps by multiple hypothesis testing and false discovery rate (FDR) control. In this way, 3D confidence maps contain separated signal from background noise in the form of local detection rates of EM density values. We demonstrate that confidence maps and FDR-based thresholding can be used for the interpretation of near-atomic resolution single-particle structures as well as lower resolution maps determined by subtomogram averaging. Confidence maps represent a conservative way of interpreting molecular structures due to minimized noise. At the same time they provide a detection error with respect to background noise, which is associated with the density and particularly beneficial for the interpretation of weaker cryo-EM densities in cases of conformational flexibility and lower occupancy of bound molecules and ions to the structure.

biophysics↗