Images taken by transmission electron microscopes are usually affected by lens aberrations and image defocus, among other factors. These distortions can be modeled in reciprocal space using the contrast transfer function (CTF). Accurate estimation and correction of the CTF is essential for restoring the high-resolution signal in cryogenic electron microscopy (cryoEM). Previously, we described the implementation of algorithms for this task in the cisTEM software package (Grant et al., 2018). Here we show that taking sample characteristics, such as thickness and tilt, into account can improve CTF estimation. This is particularly important when imaging cellular samples, where measurement of sample thickness and geometry derived from accurate modeling of the Thon ring pattern helps judging the quality of the sample. This improved CTF estimation has been implemented in CTFFIND5, a new version of the cisTEM program CTFFIND. We evaluated the accuracy of these estimates using images of tilted aquaporin crystals and eukaryotic cells thinned by focused ion beam milling. We estimate that with micrographs of sufficient quality CTFFIND5 can measure sample tilt with an accuracy of 3° and sample thickness with an accuracy of 5 nm.

Agonists enhance receptor activity by providing net-favorable binding energy to active over resting conformations, with efficiency (η) linking binding energy to gating. Previously, we showed that in nicotinic receptors, η-values are grouped into five structural pairs, correlating efficacy and affinity within each class, uniting binding with allosteric activation (Indurthi and Auerbach, 2023). Here, we use molecular dynamics (MD) simulations to investigate the low-to-high affinity transition (L→H) at the Torpedo α−δ nicotinic acetylcholine receptor neurotransmitter site. Using four agonists spanning three η-classes, the simulations reveal the structural basis of the L→H transition where: the agonist pivots around its cationic center (‘flip’), loop C undergoes staged downward displacement (‘flop’), and a compact, stable high-affinity pocket forms (‘fix’). The η derived from binding energies calculated in silico matched exact values measured experimentally in vitro. Intermediate states of the orthosteric site during receptor activation are apparent only in simulations, but could potentially be observed experimentally via time-resolved structural studies.