From sample preparation to the refinement of high-resolution molecular structures, a course offered by the team at the Center for Research in Biology of Bacteria and Bacteriophages (CEPID B3) during the 55th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology (SBBq) immersed researchers in the practice of cryo-electron microscopy, or cryo-EM – one of the most advanced techniques in structural biology. The training, held from May 17 to 19, focused on presenting, in an integrated manner, both the fundamentals and applications of cryo-EM, which is used to reveal, in detail, structures thousands of times smaller than a bacterium.

On the first day of the activity, titled “The molecular revolution: an introduction to single-particle analysis by cryo-EM”, Germán Sgro (USP-RP) introduced the proper procedures for sample preparation, covering both cryo-microscopy and transmission electron microscopy techniques with negative staining, thereby establishing the foundation for the subsequent stages.

The second session, led by Leonardo Talachia Rosa (Unicamp), was dedicated to guidance on data acquisition and initial processing. Using a tutorial dataset, participants were given remote access to the CryoSPARC software – used to process and analyze images of biomolecular particles and, from them, reconstruct high-resolution three-dimensional structures. The course made use of the computational resources of the Da Vinci server, located at the Institute of Biomedical Sciences at USP and affiliated with CEPID B3. “We had support from Professor Robson Francisco de Souza and collaborating researcher Celso Calomeno. It was essential to making everything work smoothly”, said Rosa. According to Raphaela Machado, a PhD student at CEPID B3 and course participant, these tools enabled a truly hands-on experience. “We were actually able to carry out the processing, it wasn’t just theoretical”, she explained. “We imported micrographs, performed preprocessing with motion correction and CTF estimation, as well as particle picking and extraction”, she added.

The third and final session focused on more advanced stages of the analysis, including 2D and 3D classification, model refinement, and post-processing. For the PhD student, this hands-on approach was one of the highlights of the course. “It was very interesting to follow each step while executing it in the software, understanding the importance of every stage”, she said.

By the end of the course, 100% of participants reported feeling capable of planning, at least at an initial level, the steps required to obtain a structure using cryo-EM, while 85.7% stated that they had learned how to use the CryoSPARC software throughout the training. These results reinforce CEPID B3’s role in training highly qualified researchers and in strengthening expertise in cutting-edge techniques, which are increasingly essential for the investigation of biomolecular structures and for the advancement of science in Brazil.