The ability of most cell lineages to defend themselves individually against infection can be considered as the most ancestral form of defense against pathogen pressure. We are interested in the intriguing interplay of autophagy and intracellular bacteria, where the host employs autophagy for bacterial clearance while some pathogens re-direct autophagy as a pro-survival strategy. We work towards understanding the mechanisms of restricting intracellular pathogens by studying three-dimensional structures of the protein machinery involved in this form of cytosolic host defense.
Intracellular trafficking is characterized by dynamic membrane remodeling events. Pathogens hijack autophagy and other intracellular membranes to subvert immune recogonition, replicate and to escape from host cells. We currently lack a detailed view of how the pathogen protein machinery assembles on membranes to remodel its replicative compartments. The overarching goal of this research program is to understand the structures and mechanism of these protein machineries, and those of the host involved in restricting intracellular pathogens. We use nanotechnology alongside biochemistry and cell biology to build reconstituted systems that we can manipulate precisely, and we use electron imaging methods to visualize them in molecular detail.
We actively develop new methods and technology to support our research.
Sample preparation methods for cryo-EM:
In collaboration with Andreas Engel we are developing a novel cryo-EM sample preparation method to selectively target subcellular structures for direct vitrification. Together with Jacob Hoogenboom we are working on an integrated system for correlative light and electron microscopy to aid targeting and preparation of cryo-lamellae for cellular tomography.
Density interpretation and model building:
We develop methods to facilitate interpretation of cryo-EM density maps and improve the accuracy of models derived from them. We have introduced the concept of local sharpening to optimize contrast in maps with local resolution variation and adaptive restraint weigthing for atomic model refinement against a single map target. Our efforts in improving density interpretation methods are continued as part of the CCP-EM project.
Microdiffraction: We have developed protocols to utilize the spontaneous protein crystallization in yeast peroxisomes as a source of nanocrystals for in cellulo diffraction using XFELs and electron diffraction (infamously termed “microED”) and demonstrated that collection of diffraction data is possible from protein crystals in live cells. We now focus on the development of smarter data acquisition strategies for beam-sensitive (biological) materials. Stef Smeets is developing tools for automated collection of electron diffraction data.
Kavli Institute for Nanoscience Delft
Delft University of Technology
Jacob Hoogenboom, Department of Imaging Physics
TU Delft AI Initiative (IRIS), Imaging Physics and Delft Center for Systems and Controls
Thierry Soldati, Université de Genève, Geneva (CH)
Terje Johanson, UiT – The Arctic University of Norway, Tromsø (NO)
Tom Burnley, CCP-EM