Cryo-EM refinement and model building of protein-RNA complexes

Martina Zanova, David Zapletal, Karel Kubicek, Matyas Pinkas, Jiri Novacek, Richard Stefl

Masaryk University


Cryo-electron microscopy has proven to be a powerful and innovative technique in the field of structural biology. It has gained well-deserved popularity among the scientific community due to the relative ease of sample preparation, the ability to capture the molecules under study in different conformational states, and the ability to reconstruct their structure in a matter of days. Thus, it is not surprising that the cryo-EM field and technology are growing rapidly. Many different programs can be used in the workflow of the cryo-EM data processing and the researchers should be able to choose and use them correctly according to the unique requirements of the collected dataset. In presented work, we have studied mouse RNase III in complex with double-stranded RNA molecule using cryo-EM. The cryo-EM density maps have been obtained in relatively high resolution (locally up to 3.5 Å resolution) thanks to the advanced workflow of data processing. The refinement software cryoSPARC and RELION were used to reconstruct the electron map as 3D heterogeneous refinement and local refinement of individual protein domains had a significant effect on the final resolution of the electron map. To build protein-RNA models, we used software Coot and the final structural refinement was performed using programs PHENIX and Isolde. My poster presentation highlights the latest advances in cryo-EM data processing and their direct usage in refinement of protein-RNA complexes.