Single-particle cryo-EM using alignment by classification (ABC): the structure ofLumbricus terrestrishaemoglobin

Abstract

Single-particle cryogenic electron microscopy (cryo-EM) can now yield nearatomic resolution structures of biological complexes. However, the referencebased alignment algorithms commonly used in cryo-EM suffer from reference bias, limiting their applicability (also known as the 'Einstein from random noise' problem). Low-dose cryo-EM therefore requires robust and objective approaches to reveal the structural information contained in the extremely noisy data, especially when dealing with small structures. A reference-free pipeline is presented for obtaining near-atomic resolution three-dimensional reconstructions from heterogeneous ('four-dimensional') cryo-EM data sets. The methodologies integrated in this pipeline include a posteriori camera correction, movie-based full-data-set contrast transfer function determination, movie-alignment algorithms, (Fourier-space) multivariate statistical data compression and unsupervised classification, 'random-startup' three-dimensional reconstructions, four-dimensional structural refinements and Fourier shell correlation criteria for evaluating anisotropic resolution. The procedures exclusively use information emerging from the data set itself, without external 'starting models'. Euler-angle assignments are performed by angular reconstitution rather than by the inherently slower projection-matching approaches. The comprehensive 'ABC-4D' pipeline is based on the two-dimensional referencefree 'alignment by classification' (ABC) approach, where similar images in similar orientations are grouped by unsupervised classification. Some fundamental differences between X-ray crystallography versus single-particle cryo-EM data collection and data processing are discussed. The structure of the giant haemoglobin from Lumbricus terrestris at a global resolution of $3.8 Å is presented as an example of the use of the ABC-4D procedure.

A revised order of subunits in mammalian septin complexes

“…Images of high‐salt (500 mM) samples incubated with mouse monoclonal anti‐SEPT5 were taken on a Talos F200C (Thermo Fisher Scientific) microscope at 200 kV and were also recorded using a Ceta camera with a pixel size of 2.6 Å. Imagic4D software (Image Science, Germany) was used for image processing (van Heel et al, ). Class averages of selected particles were computed after alignment by classification and subsequent rounds of multivariate statistical analysis (MSA) using selected masks (Afanasyev et al, ; van Heel, Portugal, & Schatz, ). For better visualization of the MBP location, due to the flexible linker, one of the masks used was constructed as a rectangle on the hexameric complex, so that only information from the MBP was used for classification.…”

New tools for the analysis and validation of cryo-EM maps and atomic models

“…The FSC values can be calculated either with the whole map or with one of the half-maps (maps reconstructed independently each using half of the experimental data) depending on the specific goal (see, for example, DiMaio et al, 2009;Brown et al, 2015). The FSC curve has a characteristic shape, the intersection of which with a threshold (0.143 or 0.5; Rosenthal & Henderson, 2003;van Heel & Schatz, 2005) provides the d FSC value used nowadays; however, alternative interpretations exist Afanasyev et al, 2017).…”

Repositioning septins within the core particle

“…were taken on a Talos F200C (Thermo Fisher Scientific, USA) microscope at 200 kV and were also recorded using a Ceta camera with a pixel size of 2.6 Å. Imagic4D software (Image Science, Germany) was used for image processing [27]. Class averages of selected particles were computed after alignment by classification and subsequent rounds of multivariate statistical analysis (MSA) using selected masks [28,29]. For better visualization of the MBP location, due to the flexible linker, one of the masks used was constructed as a rectangle on the hexameric complex, so that only information from the MBP was used for classification.…”