Guidelines for using Bsoft for high resolution reconstruction and validation of biomolecular structures from electron micrographs

Abstract

Cryo-electron microscopy (cryoEM) is becoming popular as a tool to solve biomolecular structures with the recent availability of direct electron detectors allowing automated acquisition of high resolution data. The Bsoft software package, developed over 20 years for analyzing electron micrographs, offers a full workflow for validated single particle analysis with extensive functionality, enabling customization for specific cases. With the increasing use of cryoEM and its automation, proper validation of the results is a bigger concern. The three major validation approaches, independent data sets, resolution-limited processing, and coherence testing, can be incorporated into any Bsoft workflow. Here, the main workflow is divided into four phases: (i) micrograph preprocessing, (ii) particle picking, (iii) particle alignment and reconstruction, and (iv) interpretation. Each of these phases represents a conceptual unit that can be automated, followed by a check point to assess the results. The aim in the first three phases is to reconstruct one or more validated maps at the best resolution possible. Map interpretation then involves identification of components, segmentation, quantification, and modeling. The algorithms in Bsoft are well established, with future plans focused on ease of use, automation and institutionalizing validation.

Unique structural features of the mitochondrial AAA+ protease AFG3L2 reveal the molecular basis for activity in health and disease

“…Local resolution estimation using the "blocres" function in the Bsoft package 62 indicates that there are regions within the reconstructions that are resolved to better than the FSCreported resolutions. However, we do not observe EM density corresponding to ordered water molecules, which should be visible at better than 2.9Å resolution so we suspect that the reported FSC-based resolutions are slightly inflated, possibly due to the presence of preferred views in the final reconstruction ( Figure S1D).…”

“…Details provided in the Methods. C. Local resolution were calculated using the Bsoft package 62 , showing that the final EM-density of core AFG3L2 WB/PI shows the resolution varies from 3.1Å at the core of the complex to 3.5Å for the built regions. D. The distribution of the Euler angles assigned to the particles in the final consensus reconstruction.…”

Structural insights into ligand efficacy and activation of the glucagon receptor

“…The final subset of 266,267 particles was imported to cisTEM for 3D local refinement, resulting in a reconstruction with global resolution of 3.1Å at FSC 0.143 39 . Local resolution was determined using the Bsoft package at cutoff FSC of 0.5 40 .…”

Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir

“…After consecutive rounds of 3D refinement and Bayesian polishing of 130,386 particle projections, the final map has an indicated global resolution of 2.5 Å at a Fourier shell correlation (FSC) of 0.143. Local resolution was determined using the Bsoft package with half maps as input maps (38).…”