For decades, mechanistic understandings of how macromolecular complexes involving transcription factor proteins fulfill their functional roles under physiological conditions has been a central theme in the field of molecular and cellular biology. Over years, revolutionary technical advancements in structural and computational biology have aimed to deduce how such complexes function by determining their high-resolution three-dimensional (3-D) structural arrangements at the atomic levels. Among number of techniques used to determine such structures, the most successful techniques have been X-ray crystallography and Nuclear magnetic resonance (NMR) spectroscopy. However, each of these techniques have their limitations. For example, despite countless successes of X-ray crystallography, some of the samples have been found hard to crystallize or certain functionally relevant states may be hard to purify, and in certain cases, even when crystallization is achieved, the nature of the crystals may make structural determination hard. Similarly, despite of NMR technique having the advantage of providing unique information about dynamics and interactions, atomic structure determination by NMR is restricted to small macromolecular size. Additionally, both techniques typically require large concentrations of relatively pure sample.

In recent years, cryo-electron microscopy (cryo-EM) has become one of the most powerful tools to solve the structure of macromolecules at near-atomic resolution (Murata & Wolf, 2018; Subramaniam, Earl, Falconieri, Milne, & Egelman, 2016; Wu & Lander, 2020). This powerful technique allows direct observation on a low dose transmission electron microscope operating at liquid nitrogen temperature or below (Murata & Wolf, 2018; Turoňová, Sikora, Schürmann, et al., 2020; Wrapp, Wang, Corbett, et al., 2020; Wu & Lander, 2020; Yao, Song, Chen, et al., 2020). Unlike, X-ray crystallography, cryo-EM does not require 3-D crystals capable of high-resolution X-ray diffraction (Murata & Wolf, 2018; Turoňová et al., 2020; Wrapp et al., 2020; Wu & Lander, 2020; Yao et al., 2020). The cryo-EM method also provides an in-depth insight into the dynamic behavior of macromolecules, which is particularly useful to determine 3-D structure of large protein complexes in their near-native environment (Murata & Wolf, 2018; Subramaniam et al., 2016; Turoňová et al., 2020; Wrapp et al., 2020; Wu & Lander, 2020; Yao et al., 2020).

In recent years, the cryo-EM technique has become indispensable for the characterization of large macromolecular assemblies, which are recalcitrant to crystallization (Murata & Wolf, 2018; Subramaniam et al., 2016; Turoňová et al., 2020; Wrapp et al., 2020; Wu & Lander, 2020; Yao et al., 2020). The transformative impact of cryo-EM single-particle analysis on the structural biology field has garnered considerable attention from the scientific community (Glaeser, 2019; Turoňová et al., 2020; Wrapp et al., 2020; Wu & Lander, 2020; Yao et al., 2020). In last 10 years or so, changes in microscope and detector technologies, have resulted into increasing numbers of near-atomic resolution (in the range of 3–5 Å) structural studies using cryo-EM technique (Murata & Wolf, 2018; Subramaniam et al., 2016; Wu & Lander, 2020). These technical advancements have been immensely helpful in determining 3-D structures of large protein complexes including membrane ion channel proteins, transcription factors, and several other signaling proteins which in turn has been especially useful both for understanding molecular mechanisms underlying protein functions and the structural basis of molecular interactions (Murata & Wolf, 2018; Subramaniam et al., 2016; Wu & Lander, 2020).

The Cryo-EM technique has superseded X-ray crystallography and NMR to emerge as a popular and effective tool for structure determination in recent times (Murata & Wolf, 2018; Subramaniam et al., 2016; Turoňová et al., 2020; Wrapp et al., 2020; Wu & Lander, 2020; Yao et al., 2020). The cryo-EM analysis is particularly advantageous for proteins that are low yield and/or not stable over extended periods. This is, in part, because unlike X-ray crystallography and NMR methods, the Cryo-EM technique utilizes significantly lower quantities of sample, and the specimens can be preserved through vitrification immediately after purification (Murata & Wolf, 2018; Subramaniam et al., 2016; Wu & Lander, 2020). In recent years, a number of protein structures have been solved using cryo-EM and single-particle electron cryo-EM has become the method of choice for the determination of protein structures (Fan, Wang, Zhang, et al., 2019; Herzik Jr, Wu, & Lander, 2019; Liu, Huynh, & Yeates, 2019; Wu & Rapoport, 2021; Yi, Yu, Wang, & O'Malley, 2021). However, there are challenges in determining structure of small proteins by cryo-EM (Turoňová et al., 2020; Wrapp et al., 2020; Wu, Avila-Sakar, Kim, et al., 2012; Yao et al., 2020).

To certain extent, this has been overcome by using a fusion or scaffold approach in which the protein of interest is complexed with binding partners such as Fab fragments of antibodies, or nanobodies (Fan et al., 2019; Herzik Jr et al., 2019; Liu et al., 2019; Turoňová et al., 2020; Wrapp et al., 2020; Wu et al., 2012; Wu & Rapoport, 2021; Yao et al., 2020). Among others, nanobodies have gained significant popularity as versatile binding partners of target proteins due, in part, to their abilities to bind to small-exposed protein surfaces, to be produced in large quantities in a fairly short time, and to often lock a protein into a fixed conformation (Ahmad et al., 2021; Fan et al., 2019; Herzik Jr et al., 2019; Murata & Wolf, 2018; Turoňová et al., 2020; Wrapp et al., 2020; Wu & Rapoport, 2021; Yao et al., 2020).