Netherlands Cancer Institute
The Netherlands Cancer Institute (NKI) is an integrated cancer center with dedicated research facilities connected to the Antoni van Leeuwenhoek cancer hospital. The NKI has excellent facilities for advanced cell biology, confocal microscopy, live-cell imaging, DNA microarray technology, biophysics, high-through-put screening and structural biology. The atmosphere in the Netherlands Cancer Institute is highly interactive, with multiple inter-group work discussion weekly and several informal fora.
The group of Titia Sixma uses a combination of structural, biochemical and biophysical techniques to study protein function at the molecular level. Within the project we will use structural biology to determines structures of relevant mismatch repair complexes. The work is directed towards regulation of ubiquitin conjugation and DNA mismatch repair. Theories resulting from structural work are validated using biophysical, biochemical and cell-based approaches.
Available equipment and expertise immediately relevant to the training programme includes recombinant protein production in E.coli and insect cells, protein purification (ÄKTAfplc) and All equipment necessary for X-ray crystallography is available, including nano-robotics for crystallization (CyBi-SELMA / Mosquito / TOPAZ), imaging robotics at two temperatures (4 and 18 degrees) (Rock imager), robotics for generating optimized screens (Formulatrix). There is regular access to synchrotron beamlines at ESRF, SLS and Hamburg and to NECEN for cryo-EM. The biophysics setup includes stopped-flow fluorimeter for transient kinetics (TgK Scientific), surface plasmon resonance (Biacore T100, GE), isothermal calorimetry (MIcrocal), fluorescence plate reader (Pherastar) multi-angle laser light scattering (Whyat), microscale thermophoresis (Monolith, Nanotemper), analytical chromatography (ÄKTAmicro) and protein stability analysis (Optim, Avacta)
Role in project
A PhD student will focus on the conformational states of DNA mismatch repair proteins using structural biology, inparticular cryo-EM and X-ray crystallography. Additionally, students from other participants will be hosted for extensive secondments, to be trained in protein purification and characterization, to investigate the architecture of protein-DNA complexes using scanning force microscopy, and to set up DNA repair assays.
Prof. Dr. Titia K. Sixma, Principal Investigator and Scientific Coordinator
Dr. Alexander Fish, biophysical expert
Doreth Kok, structural biology
Herrie Winterwerp, Technical expert
Fellow: Susanne Brueckner