MRC2020: improvements to Ximdisp and the MRC image-processing programs

Abstract

The great success of single-particle electron cryo-microscopy (cryoEM) during the last decade has involved the development of powerful new computer programs and packages that guide the user along a recommended processing workflow, in which the wisdom and choices made by the developers help everyone, especially new users, to obtain excellent results. The ability to carry out novel, non-standard or unusual combinations of image-processing steps is sometimes compromised by the convenience of a standard procedure. Some of the older programs were written with great flexibility and are still very valuable. Among these, the original MRC image-processing programs for structure determination by 2D crystal and helical processing alongside general-purpose utility programs such as Ximdisp, label, imedit and twofile are still available. This work describes an updated version of the MRC software package (MRC2020) that is freely available from CCP-EM. It includes new features and improvements such as extensions to the MRC format that retain the versatility of the package and make it particularly useful for testing novel computational procedures in cryoEM.

Keywords: MRC2020; cryoEM; image-processing programs.

Figure 1

Multi-image editing from a stack of images in Ximdisp: a gallery of images can be displayed from a multi-stack file. Poor quality images can be removed manually and are marked by a cross (yellow). On completion an output stack file is written, with the marked images removed and a list of their numbers in a separate file. The illustration shows hepatitis B virus core particles (see Böttcher et al., 1997; and EMD-13734).

Figure 2

Filament boxing option of Ximdisp. The user sets the box width before selecting each end of each candidate filament. Box corners are stored in a file which can be read by a separate program for calculating box centres with a user set inter-box distance. The box centres are stored in a file which can be read directly into RELION for processing. The illustration shows HsRad51 complexed with single-stranded DNA (EMD-8183; Short et al., 2016 ▸).

Figure 3

Interactive display of Fourier transforms from helical structures in Ximdisp. In this mode, the filament is boxed (a), then vertically aligned (inset) before calculation and display (b) of the transform. It is possible to sample the quality of various parts of the filament prior to selection by moving the box and re-calculating the transform. The illustration shows an acetyl­choline receptor tube (see Unwin, 2005; and EMD-11239).

Figure 4

Picking particles from tilted pairs in Ximdisp. The specimen imaged in this example is Haliotis diversicolor hemocyanin, molecular weight 8 MDa (EMD-5585, EMD-5586; Zhang et al., 2013 ▸). Images were recorded on film at 200 keV. Ximdisp was used to pick particles manually from the left image (a) which was recorded with a specimen tilt angle of −5°. This was then transferred by Ximdisp to the middle image (b) which was recorded at a tilt angle of +5°. The positions of a few particles in the middle image were corrected manually and the shifts calculated by Ximdisp were used to correct the remaining particles automatically, as seen in the right image (c). A plot was obtained after alignment of both −5 and +5° sets of particles to the starting model and the tilt axis and angle describing the geometrical relationship between the two sets of particles calculated. The resulting tilt-pair plot is shown in Fig. 5 ▸(a).

Figure 5

Examples of tilt-pair plots from two different samples, used to validate determination of the single-particle orientations that are needed to calculate the 3D structure. In (a) the change in the angle of the Haliotis diversicolor hemocyanin particles is clustered at 10°, which was the angular difference between the two images, indicating the validity of the 3D model. The structure of this hemocyanin was solved subsequently to a resolution of 4.5 Å (Zhang et al., 2013 ▸). In (b) a similar experiment on Norwalk virus-like particles with a 5° change in tilt angle between the two images is shown. In this experiment, three pairs of images were recorded, with particles from different image pairs shown in different colours. With molecular weights of 8 and 10 MDa, respectively, these two additional tilt-pair parameter plots fill in the gap in particle size, between 3 and 50 MDa, that was missing from an earlier publication (PDB code 1ihm; Prasad et al., 1999 ▸) on a range of different specimens.