Hierarchy of Biological Complexity

1. Hierarchy of Biological Complexity LIFE Interactions of machines (molecular and cellular dynamics) Macromolecular machines Proteins and nucleic acids Sequences GENOMES

3. The ISCOMMM Team

5. Electron Cryo-microscopy and Image Analysisof Macromolecular Complexes

6. Experimental Design • Large-scale production of purified vaccinia virus particles • Isolation and purification of vaccinia molecular machines • Identification of protein composition by mass spectrometry • Generation of antibodies directed again proteins in the machine • Formation of antibody-molecular machine complexes • Preparation of frozen-hydrated specimens • Automated recording of cryo-EM images • Automated extraction of particle images from electron micrographs • Automated determination of 3D density maps • Protein localization by use of antibody labeling and difference map analysis • Validation of computational predictions of protein-protein interactions

8. Cryo-EM Structure of Integrin αIIbβ3 Brian Adair and Mark Yeager

9. Docking of X-ray Structure of αVβ3 Ectodomaininto Cryo-EM Map of αIIbβ3 Lower resolution EM maps of functionally important, transient states can be combined with high resolution X-ray structures to derive atomic models.

10. Integrin Molecular Models X-ray High affinity Low Affinity

11. Cryo-EM density fitting Protein-protein docking prediction & density correlation fitting Visual fitting

12. Cryo-EM density fitting Protein-protein docking prediction & density correlation fitting Visual fitting

13. The Scripps Research Institute and Scripps Clinic