CTER—Rapid estimation of CTF parameters with error assessment

Abstract

In structural electron microscopy, the accurate estimation of the Contrast Transfer Function (CTF) parameters, particularly defocus and astigmatism, is of utmost importance for both initial evaluation of micrograph quality and for subsequent structure determination. Due to increases in the rate of data collection on modern microscopes equipped with new generation cameras, it is also important that the CTF estimation can be done rapidly and with minimal user intervention. Finally, in order to minimize the necessity for manual screening of the micrographs by a user it is necessary to provide an assessment of the errors of fitted parameters values. In this work we introduce CTER, a CTF parameters estimation method distinguished by its computational efficiency. The efficiency of the method makes it suitable for high-throughput EM data collection, and enables the use of a statistical resampling technique, bootstrap, that yields standard deviations of estimated defocus and astigmatism amplitude and angle, thus facilitating the automation of the process of screening out inferior micrograph data. Furthermore, CTER also outputs the spatial frequency limit imposed by reciprocal space aliasing of the discrete form of the CTF and the finite window size. We demonstrate the efficiency and accuracy of CTER using a data set collected on a 300 kV Tecnai Polara (FEI) using the K2 Summit DED camera in super-resolution counting mode. Using CTER we obtained a structure of the 80S ribosome whose large subunit had a resolution of 4.03 Å without, and 3.85 Å with, inclusion of astigmatism parameters.

Structural Snapshots of Actively Translating Human Ribosomes

“…Although it has been demonstrated that near-atomic resolution maps for relative invariant parts of ribosome can be obtained, e.g. by focusing the refinement on the large ribosomal 60S subunit (Penczek et al, 2014), and composite near-complete atomic models of the eukaryotic ribosome can be constructed by combining the best resolved parts from different functional states (Voorhees et al, 2014), we tried to represent distinct complexes by a single cryo-EM map each. This was to ensure that we describe distinct functional states and are able to faithfully visualize structural links between remote functional sites (Agmon et al, 2005), e.g.…”

Structure of the F-actin–tropomyosin complex

“…Thus, the approximate distance between them (~32 Å) slightly exceeded the rise of the helical assembly of actin (~27–28 Å), as required by the helicon design. The defocus and astigmatism and the accuracy of both were determined by cter 35 in SPARX. All segments were aligned and classified using reference-free alignment and k -means classification procedures implemented in SPARX (Extended Data Fig.…”

“…Once the refined map reached a resolution of 6.5 Å, as determined by the FSC 0.5 criterion 36 , we masked out tropomyosin in the reference volume before continuing with high-resolution refinements of F-actin. In the last refinement iteration, we used only filaments derived from summed up frames with a drift of less than 7 Å, as estimated in the motion correction 34 step and with a sufficient content of high-resolution information, as determined by cter 35 in SPARX. This yielded a subset of 74,228 segments for the final map, which corresponded to ~120,000 asymmetric subunits.…”

Studies on the X-Ray and Solution Structure of FeoB from Escherichia coli BL21

“…The pixel-size at the object plane corresponds to 2.9 Å /pixel. Defocus estimation was performed using CTER (23), and micrographs were preselected based on calculated defocus and astigmatism values. Strong heterogeneity precluded particle classification procedures.…”