EM studies on proteins

Abstract

In this study we studied various proteins and protein complexes by means of Single Particle Analysis (SPA), a 3D Electron Microscopy (EM) technique. High resolution images were collected with cryo TEM; lower resolution tests were imaged with negative stain TEM. In Part I (carried out in Friedrich Fo¨rster’s Cryo EM group at Utrecht University in close collaboration with Piet Gros’ Crystal and Structural Chemistry group) we study a complement protein complex XYZ to assess where Protein Z binds to and/or cleaves XY. We find many particles in the X and XY classes, which were reconstructed to 8.7 and 7.6 ˚A respectively, the full complex is not detected in the dataset. Both Relion and CryoSparc were used for the reconstruction, as well as Gautomatch, Eman2 and Chimera. Ongoing work in the group has improved this reconstruction, however not included in this dissertation. In Part II (carried out in the Structural & Biophysics Sciences group at GSK R&D UK), apart from a training data set of apoferritin, we studied another protein complex BCD+ of 190 kDa. Also here, both complex and individual protein were found. Protein D (150kDa), which formed the major part of the data, had one or more moving domain(s). A general protocol for negative stain and cryo EM processing has been established. A protocol for Tm buffer screening has also been established to optimize stability of the protein in question. During this part of the project, CryoSparc has not been used.