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.
We mostly use publicly available data sets from the EMPIAR data base at EMBL-EBI. For this study, we have submitted to this data base our own data on the human gamma-secretase complex (EMPIAR-10194) and on the high-resolution apo-ferritin sample described in the text (EMPIAR-10200).
An atomic structure of human gamma-secretase [2925 multi-frame micrographs composed of 20 frames each in MRCS format]Electron Microscopy Public Image Archive, EMPIAR-10194.
Human apo-ferritin reconstructed in RELION-3.0Electron Microscopy Public Image Archive, EMPIAR-10200.
2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitorElectron Microscopy Public Image Archive, EMPIAR-10061.
40 Degree Tilted Single-Particle CryoEM of Highly Preferred Orientated Influenza Hemagglutinin TrimerElectron Microscopy Public Image Archive, EMPIAR-10097.
Bacteriophage P22 mature virion capsid proteinElectron Microscopy Public Image Archive, EMPIAR-10083.
- Sjors HW Scheres
- Jasenko Zivanov
- Erik Lindahl
- Erik Lindahl
- Takanori Nakane
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Edward H Egelman, University of Virginia, United States
© 2018, Zivanov et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and six different sites of the hexamer protein Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology.
In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2’s nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B’s β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.