GTL FacilitiesCharacterization and Imaging of

Molecular Machines

Lee Makowski

2

Genomes to Life Facilities for 21st 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

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

Facility 3: Characterization and Imaging of Molecular Machines

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

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

Identifying the parts of a molecular machine

Is the first step to understanding

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

Detailed images will be needed to complete the linkage of structure to function

Zhou et al., Science 2000

300 keV cryoEM

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

Developing models for the assembly, disassembly and function of molecular machines will be

computationally challenging

Machines

Proteins

Cellular systems

11

Facility IV Cellular Systems

Gene Sequence

Gene Sequence

High Validity DataResource for

Biological Community

High Validity DataResource for

Biological Community

Facility III: Characterization and Imaging of Molecular Machines

Facility IIProteome

Data

Facility IReagents

Microbe Growth

Complex Isolation

Data Interpretation/

Archival, Modeling

Image Complexes

BiophysicalCharacterization

ComponentIdentification