Jürgen Plitzko, PhD.
THEMATIC RESEARCH FOCUS
- Structure and function of macromolecular complexes and cellular assemblies.
- Molecular mechanisms of virus replication and host-pathogen interactions.
- Time-resolved electron microscopy of biological processes at millisecond timescale.
- Investigate the structure, interactions and conformational dynamics of macromolecular complexes and assemblies to understand the structural basis of their function and regulation.
- Elucidate the structural and conformational features of viral capsids to understand the molecular mechanisms of virus assembly, maturation and interaction with the host cell.
- Develop and apply new methods of time resolved cryo-electron microscopy to study assembly and conformational dynamics of transient macromolecular complexes.
CONTENT OF RESEARCH
We utilize cryoelectron microscopy (cryoEM) and 3-D image reconstruction as the main tool to characterize the structural organization, dynamics and interactions of large macromolecular complexes and assemblies, such as the ribosome, viral capsids or filaments of the cytoskeleton. We image these complexes as whole units of action at distinct functional states and determine the structural and mechanistic features of their function. We employ methods of single particle analysis and electron tomography to determine structural information about purified complexes and assemblies in vitro. To gain insight into molecular processes inside eukaryotic cells, we use unique techniques of focused ion beam micromachining to prepare thin sections (lamellas) through vitrified cells that are then suitable for electron tomography. Additionally, we develop methods of time-resolved cryoEM at the millisecond time scale to capture structural and mechanical features of fast biological processes.
KEY RESEARCH EQUIPMENT
High-end electron microscope for high-resolution cryoEM and cryo-tomography (FEI Titan Krios) equipped with an energy filter and a direct detector camera, conventional cryoEM microscope (FEI F20) equipped with a CCD camera and a dual beam FIB/SEM instrument (Versa3D) for micromachining of thin lamellas of vitrified cells usable for electron tomography.
Biochemical laboratory equipped for purification and characterization of proteins and nucleic acidsn and their complexes. Instrumentation for preparation of vitrified specimen for electron cryo-microscopy (Vitrobot Mark IV).
Current projects include these topics:
1) Structural studies of large regulatory complexes in cellular signaling pathways:
The TSC1-TSC2 complex is part of the target-of-rapamycin signaling pathway. It integrates signals about nutrient and stress conditions of the cell and conveys this information downstream to two distinct TOR complexes that are central to this signaling pathway: (i) mTORC1, which regulates cell metabolism via activation of several downstream kineses (e.g. S6K or Akt) and (ii) mTORC2, which regulates organization of actin filaments in cell division. Mutations that deactivate the TSC1-TSC2 complex lead to a multisystemic tumor syndrome called tuberous sclerosis complex. We aim to determine the structural features of the TSC1-TSC2 complex and elucidate its function in activation of the mTOR complexes.
2) Assembly, maturation and cell entry mechanisms of large dsDNA viruses:
Nucleocytoplasmic large DNA viruses belong to the largest and most complicated viruses known that replicate in the cell nucleus and cytoplasm with only little help from the host replication machinery. Here, we focus on ranaviruses from the family Iridoviridae that have caused large economic losses in aquaculture industry and currently contribute to dramatic declines of amphibian species worldwide. Ranaviruses have multilayered virions with an icosahedral proteinaceous capsid and an internal membrane that separates the DNA core from the capsid shell. The capsid is first assembled as a DNA-free procapsid that is subsequently filled with the viral genome. A minor fraction of progeny virions buds from the plasmatic membrane and contains an external envelope. Both naked capsids and enveloped virions are infectious with probably different modes of cell entry. We aim to elucidate the structural basis for capsid assembly, maturation and cell entry of these giant viruses.
3) Time-resolved cryo-electron microscopy of the ribosome:
The ribosome is the site of protein synthesis in the cell. The 30S and 50S subunits of the E. coli 70S ribosome are held together by at least 12 intersubunit contacts. These contacts include RNA-RNA, RNA-protein, and protein-protein interactions. We have developed and implemented a class of microfluidic devices that mixes two components to completion within 0.4 ms and sprays the mixture in the form of microdroplets onto an EM grid that is being plunged into cryogen. The total reaction time before cryo-fixation can be as short as 9.4 ms. We have used devices with reaction times of 9.4 and 43 ms to study the association between the 30S and 50S subunits into 70S ribosomes. We have shown that we are able to trap intermediates in the association of these two subunits, where a subset of the 12 intersubunit bridges have not yet formed (manuscript in preparation). In vivo, the formation of the 70S ribosome is more intricate, requiring in addition an initiator tRNA, mRNA, and three initiation factors. Biochemical studies show steps in this pathway with reaction times accessible by our microfluidic devices. One of our aims is to capture intermediates in the initiation pathway.
- Rigort A, Villa E, Bäuerlein FJ, Engel BD, Plitzko JM. (2012) Integrative approaches for cellular cryo-electron tomography: correlative imaging and focused ion beam micromachining. Methods Cell Biol. 111, 259-281.
- Rigort A, Bäuerlein FJ, Villa E, Eibauer M, Laugks T, Baumeister W, Plitzko JM. (2012) Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc. Natl. Acad. Sci. USA. 109, 4449-4454.
- Nemecek, D., Qiao, J., Mindich, L., Steven, A.C., Heymann, J.B. (2012) Packaging accessory protein P7 and polymerase P2 have mutually occluding binding sites inside the bacteriophage ϕ6 procapsid. J. Virol., 86, 11616.
- Yokoyama, T., Shaikh, T.R., Iwakura, N., Kaji, H., Kaji, A., Agrawal, R.K. (2012) Structural insights into initial and intermediate steps of the ribosome-recycling process. EMBO J. 31, 1836-1846.
- Nemecek, D., Cheng, N., Qiao, J., Mindich, L., Steven, A.C., Heymann, J.B. (2011) Stepwise Expansion of the Bacteriophage ϕ6 Procapsid: Possible Packaging Intermediates. J. Mol. Biol., 414, 260-271.
- Plitzko JM, Rigort A, Leis A. (2009) Correlative cryo-light microscopy and cryo-electron tomography: from cellular territories to molecular landscapes. Curr Opin Biotechnol. 20, 83-89.
- Shaikh, T.R., Barnard, D., Meng, X., Wagenknecht, T. (2009) Implementation of a flash-photolysis system for time-resolved cryo-electron microscopy. J. Struct. Biol. 165, 184-189.