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Review
. 2016;579:159-89.
doi: 10.1016/bs.mie.2016.05.001. Epub 2016 Jul 1.

Single-Particle Refinement and Variability Analysis in EMAN2.1

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Free PMC article
Review

Single-Particle Refinement and Variability Analysis in EMAN2.1

S J Ludtke. Methods Enzymol. .
Free PMC article

Abstract

CryoEM single-particle reconstruction has been growing rapidly over the last 3 years largely due to the development of direct electron detectors, which have provided data with dramatic improvements in image quality. It is now possible in many cases to produce near-atomic resolution structures, and yet 2/3 of published structures remain at substantially lower resolutions. One important cause for this is compositional and conformational heterogeneity, which is both a resolution-limiting factor and presenting a unique opportunity to better relate structure to function. This manuscript discusses the canonical methods for high-resolution refinement in EMAN2.12, and then considers the wide range of available methods within this package for resolving structural variability, targeting both improved resolution and additional knowledge about particle dynamics.

Keywords: 3-D reconstruction; CryoEM; EMAN; Heterogeneity; Image processing; Motion; Single-particle analysis; Structural biology; Structural variability.

Figures

Figure 1
Figure 1
A diagram showing how e2refine_split separates a single 3-D refinement into two distinct maps, by subclassifying the particles within each class-average.
Figure 2
Figure 2
A diagram showing how particles can be accurately classified between two 3-D reference maps. The classification performed using this process provides demonstrably better statistical separation than standard multimodel refinement (4.2), provided the major differences between the two references are fairly localized.
Figure 3
Figure 3
A diagram showing how local fragments can be accurately isolated from 2-D images of individual particles based on a 3-D mask and a refinement of the complete data set.
Figure 4
Figure 4
A panel showing just a few of EMAN2.1’s many graphical tools. The main project manager is shown in the upper right, with a portion of the dialog for a run of e2refine_easy shown. On the upper left is the file browser, note the metadata displayed in the columns of the browser, as well as the actions available at the bottom of the window specific to the selected file type. The bottom three windows show e2filtertool operating on a 3-D volume. The 3-D display in the center shows an isosurface with a slice through the middle and a 3-D arrow annotation. The control panel, which permits modifying this display, is shown on the left. On the right is the e2filtertool dialog, where a sequence of two image processing operations have been added: first, a “local normalization” operation, which helps compensate for low resolution noise and ice gradients; second, a Gaussian low-pass filter set to 8 Å resolution. The parameters of these operations can be adjusted in real-time with corresponding updates in the 3-D display. 2-D images and stacks can also be processed using this program. Any of over 200 image processing operations can be used from this interface, and command-line parameters are provided mimicking the final adjusted parameters.

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