Structural biology has catered to the atomic resolution structure of biological macromolecules, facilitating the mechanistic understanding of their biological functions (Curry, 2015). Conventional structural biology techniques, such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, profoundly influenced the translational research (Curry, 2015, Congreve et al., 2005), whereas cryo-EM is an emerging tool in the realm of structural biology. The utilization of cryo-EM has been significantly enhanced by the latest developments in detector technology, rendering it an indispensable tool for studying very large macromolecular moieties at near-atomic resolution (Yip et al., 2020, Sente et al., 2020). The application of cryo-EM in this framework is evidenced by the recent achievement of an atomic-resolution structure for molecules with sizes ranging from approximately 200 kDa to megadaltons (Yip et al., 2020, Sente et al., 2020, Macé et al., 2022, Fromm et al., 2023, Chari and Stark, 2023).

Although the technique has made significant strides in recent years toward elucidating the structure of higher molecular mass macromolecules, single particle cryo-EM is still not considered amiable for achieving higher resolution structures for smaller (below 100 kDa) macromolecules (Chari and Stark, 2023, Renaud et al., 2018). Due to the poor signal-to-noise ratio (SNR) the accurate particle alignment is impeded, which lowers the achievable resolution of 3D reconstructed map (Chari and Stark, 2023, Renaud et al., 2018). So far, cryo-EM has demonstrated limited success in resolving the structures of small macromolecules, as evidenced by the relatively low number of structures for these entities (Supplementary Table S1).

In recent years, the objective of investigating small macromolecules through cryo-EM has been the subject of extensive research, encompassing various methodologies and novel advancements. The volta phase plate (VPP) has been successfully used to determine the cryo-EM structure of Haemoglobin (64 kDa) and Streptavidin (52 kDa), both at a resolution of 3.2 Å (Fan et al., 2019, Khoshouei et al., 2017). Various protein scaffolds, such as modified ankyrin repeat proteins (DARPins) (Liu et al., 2019), nanobodies (Wu and Rapoport, 2021) and Fab fragments of antibodies (Wu et al., 2012) have contributed to success in determining the structural details by increasing the overall size of the target small macromolecules. Despite being regarded as advanced techniques, VPP and protein scaffolds have certain practical limitations. For instance, VPP can result in the loss of scattered electrons from the specimen and necessitates precise focusing of the specimen and alignment of VPP to the back focal plane (Wang and Fan, 2019). Likewise, establishing a functional protein scaffold system and generating nanobodies/Fab fragments against a particular target protein necessitates substantial bio-engineering and extensive library screening efforts, thereby limiting their broader application (Liu et al., 2019, Wu and Rapoport, 2021). Therefore, it is imperative for further advancements to establish a universal application of cryo-EM in structural biology.

More recently, the conventional single particle cryo-EM has made significant advancements, primarily due to the establishment of robust data processing softwares (Scheres, 2012, Punjani et al., 2017). The cryo-EM structures of small proteins such as 41 kDa maltose binding protein (EMD-39117) and 52 kDa streptavidin (EMD-31083) were determined at a near-atomic resolutions of 2.3 Å and 1.7 Å, respectively. However, there is still a limited information regarding comprehensive data processing strategies for small proteins. Here, we report the cryo-EM structure of the N-terminal domain (NTD) of FKBP53 from Arabidopsis thaliana, a 55 kDa pentameric protein, resolved at 2.0 Å nominal resolution. We have systematically documented the image processing workflow that yielded high-resolution structural details of this small biological macromolecule using single particle cryo-EM.