1. GTL Facilities Characterization and Imaging of Molecular Machines Lee Makowski

2. 2 Genomes to Life Facilities for 21 st Century Biology Facility III Characterization and Imaging of Molecular Machines Isolate the repertoire of molecular machines. Characterize machines in terms of composition and molecular organization. Facility III Facility I Facility IV Facility II

3. 3 Nearly every major process in a cell is carried out by assemblies of 10 or more protein molecules Identifying the complexes and understanding their function represents a huge challenge Facility 3: Characterization and Imaging of Molecular Machines

4. 4 GOAL: Isolation, identification and characterization of 1000 ’ s of molecular machines; providing revolutionary new information and capabilities to the biological community Repertoire of protein complexes Biophysically characterize complexes Develop principles, theory and predictive models Understanding how molecular machines operate at the molecular level will unlock the capability to control useful biochemical processes in a microbe and apply them to DOE mission needs.

5. 5 Will Require State-of-the-Art Analytical and Computational Tools Automated molecular machine stablization/isolation techniques High performance mass spectrometry Scattering techniques— optical, neutron, x-ray Imaging tools-cryoEM, optical, force microscopy Bioinformatics tools and databases/data analysis tools Modeling and simulation

6. 6 Identifying the parts of a molecular machine Is the first step to understanding

7. 7 Determining the relative positions of gene products in a machine can provide important clues to their functions Yeast spindle pole body - microtubule organizing center Novel imaging tools will be needed to characterize the relative positions of each one

8. 8 Detailed images will be needed to complete the linkage of structure to function Zhou et al., Science 2000 300 keV cryoEM

9. 9 Interaction Mapping and Imaging are Computationally Intensive For each organism: thousands of gene products tens of thousands of intermolecular interactions For each complex: thousands of images studied under different conditions localize each component data in three dimensions … and untold numbers of molecular complexes

10. 10 Developing models for the assembly, disassembly and function of molecular machines will be computationally challenging Machines Proteins Cellular systems

11. 11 Facility IV Cellular Systems Gene Sequence High Validity Data Resource for Biological Community High Validity Data Resource for Biological Community Facility III: Characterization and Imaging of Molecular Machines Facility II Proteome Data Facility I Reagents Microbe Growth Complex Isolation Data Interpretation/ Archival, Modeling Image Complexes Biophysical Characterization Component Identification