Seminar: Peter Peters

Beauty and benefits of cryo-EM for research on nano machines

Date:
3 February 2017 10:00 hrs. - 11:00 hrs.
Location:
Figdor Lecture Theatre, 8th floor RIMLS Building, Geert Grooteplein 26-28, route 289
Title:
Beauty and benefits of cryo-EM for research on nano machines
Speaker(s):

Prof. Peter Peters, The Maastricht Multimodal Molecular Imaging Institute (M4I)

Host(s):

Prof. Carl Figdor, Dept. of Tumor Immunology, RIMLS

03-02-2017 10:00:0003-02-2017 11:00:00Europe/AmsterdamBeauty and benefits of cryo-EM for research on nano machines Figdor Lecture Theatre, 8th floor RIMLS Building, Geert Grooteplein 26-28, route 289Rimlsrimls@radboudumc.nl

Remarks / more information:

undefinedWe found that after prolonged infection in macrophages and dendritic cells, M. tuberculosis translocates from phago-lysosomes to the cytosol and kills the host cell a few days later, while the BCG vaccine strain fails to translocate (Cell; 129:1287). This process is dependent on a gene in the extended RD1 region (ext-RD1). As such, further research focused on BCG with a knock-in of the entire ext-RD1, which was recently identified by a former team member as a novel type VII secretion system (T7SS) (Abdallah AM et al., Nat. Rev. Microbiol. 5:883). The main result is that these bacteria also translocate to the cytosol of the host cell. Thus, the ESX-1 secretion system is sufficient for translocation (Abdallah AM et al., J. Immunol. 2187:4744 and Houben et al., Cell Microbiol. 14:1287).

Since my move to Maastricht University, where I was invited to establish a new Institute for Nanoscopy (www.maastrichtuniversity.nl/m4i), we use cryo-EM single-particle analysis (SPA) and cryo-EM tomography to investigate recombinant purified proteins of individual gene products from the T7SS. In addition, we are purifying the entire intact T7SS structure using biochemical methods for 3D reconstruction. We will also generate lamellae of infected cells using cryo-FIB/SEM technology. The workflow starts with a high precision of localization light microscope (CorrSight), with live cell imaging of cell cultures on an EM carrier, which can be used throughout the complete workflow in order to prevent loss of orientation and sample contamination or  destruction. The instrument is a unique and dedicated light microscope for correlative microscopy, which includes a fully integrated spinning disk system. Once a specific event or location of a GFP-tagged protein of the T7SS has been identified by light microscopy and preserved by cryo-fixation, the vitrified sample is re-examined for GFP expression at 50-nm precision in a dedicated cryo-stage. This is done in order to precisely determine, in 3D, the position of interest. After transferring the sample to the cryo-FIB/SEM DualBeam (Scios), the identified region is thinned down to approximately 150 nm without artifacts, using the focused ion beam. All data, such as positions of interest and recorded images, from the LM is transferred to the cryo-FIB/SEM DualBeam by correlative software (MAPS) in order to know exactly where (to 50 nm accuracy) to prepare the lamella for subsequent high-resolution cryo-TEM imaging. After thinning the grids, they are loaded in the autoloader and transferred to the cryo-TEM (Arctica or Krios) for high-resolution cryo-tomography. The Arctica is equipped with a phase plate, a highly sensitive Falcon III direct electron detector for low-dose imaging and tomography software for automated low-dose tomography. The cryo-SPA, X-ray, and NMR data of the T7SS can then be docked onto the images from vitreous sections or lamellae to construct a macromolecular map of the tubercle bacillus within the host cell. The broader objective is to gain insight into the structure and function of the mechanism for T7SS-mediated translocation. This knowledge should lay the groundwork for the development of novel antibiotics and better vaccines. Setting up this lab and training 15 new people required more than two years of intense work. Now we are starting to generate exciting data. I would like to share this endeavor since with the emerging “revolution in resolution” of the cryo-EM field many other institutions are initiating similar initiatives.

Key Publications

  • Subcellular distribution of the prion protein in sickness and in health. Virus Research, 207, 136-145, 2015
  • A posteriori correction of camera characteristics from large image data sets. Scientific Reports, 5, 2015
  • In Vitro Expansion of Human Gastric Epithelial Stem Cells and Their Responses to Bacterial Infection. Gastroenterology, 148, 126-136, 2015

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