High-speed macromolecular structure determination on a Superbend Beamline 8.3.1

1. High-speed macromolecular structure determination on a Superbend Beamline 8.3.1 J.M. Holton1, C. Chu2, K. Corbett2, J. Erzberger2, R. Fennel-Fezzie2, J. Turner3 , D. Minor3 , R.J. Fletterick3 , J.M. Berger2, T.C. Alber2 1Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA, 2University of California, Berkeley, CA, 3University of California, San Francisco, CA The work was performed at the Advanced Light Source of Lawrence Berkeley National Laboratory, which is operated byDepartments of Energy’s Office of Basic Energy Science with Contract No. DE-AC03-76SF00098. Superbend Protein Crystal (preserved at 90K in nylon loop) Atomic Model (1000-1,000,000 atoms) Drug Discovery Elves Understanding Disease Torroid mirror MOSFLM SCALA TRUNCATE SCALEIT SHELX SOLVE MLPHARE DM ARP_WARP REFMAC Si(111) monochromator Parabolic mirror New insights Diffraction Images (~1000) Protein design Protein motors Chromasome condensation DNA replication initiation DNA topology Electron density at 1.5Å from a designed protein. This new protein was conceived using structural information from dozens of natural proteins. The high resolution structure validates our understanding of how natural proteins specify their structures. This map was obtained five hours after the data collection began. MCAK protein strongly resembles motor proteins that crawl along microtubules (kinesins). However, MCAK actively depolymerizes microtubules in the kinetochore. The structure of MCAK helps us understand how similar structures can have radically different functions. The structure of this bacterial DNA Replication initiation protein (DnaA) suggests a common structural theme in replication initiation across all kingdoms of life. This structure was solved to 2.7Å resolution at ALS Beamline 8.3.1 in less than one hour. Primase (DnaG) proteins initiate the DNA replication process in all forms of life. This S. aureus primase was solved to 1.8Å resolution at 8.3.1 and illustrates the high degree of conservation in the structure of this molecule in every living thing. Electron density at 2.5Å from a DNA topoisomerase subunit. This enzyme untangles DNA molecules during replication. This section of density highlights an isolated -helix. Electron density at 3.5Å from a bacterial chromosome condensation and segregation protein. Two -helices are apparent and two selenium atom positions are shown in green. This initial map was obtained less than one hour after the data collection began.

2. What is Protein?

3. What is Protein? • 50% (dry weight) of cells

4. What is Protein? • 50% (dry weight) of cells • ~30,000 different kinds in humans

6. What is Protein? • 50% (dry weight) of cells • ~30,000 different kinds in humans

7. What is Protein? • 50% (dry weight) of cells • ~30,000 different kinds in humans • Large molecules (1000-1000000 atoms)

8. What is Protein? • 50% (dry weight) of cells • ~30,000 different kinds in humans • Large molecules (1000-1000000 atoms) • Incredibly well-organized

9. What is Protein? • 50% (dry weight) of cells • ~30,000 different kinds in humans • Large molecules (1000-1000000 atoms) • Incredibly well-organized • All 30,000 necessary for life

10. What do Proteins do?

11. What do Proteins do? • Break down food

12. What do Proteins do? • Break down food • Build new molecules

13. What do Proteins do? • Break down food • Build new molecules • Hold cells together

14. What do Proteins do? • Break down food • Build new molecules • Hold cells together • Move objects

15. Aspartate Transcarbamoylase

16. Proteins Move

17. How do you get the structure?

18. How do you get the structure? • Purify the protein

19. How do you get the structure? • Purify the protein • Crystallize it

20. How do you get the structure? • Purify the protein • Crystallize it • Record x-ray diffraction patterns

21. How do you get the structure? • Purify the protein • Crystallize it • Record x-ray diffraction patterns • Calculate electron density

22. How do you get the structure? • Purify the protein • Crystallize it • Record x-ray diffraction patterns • Calculate electron density • Build an atomic model

23. How do you get the structure? • Purify the protein • Crystallize it • Record x-ray diffraction patterns • Calculate electron density • Build an atomic model

24. Protein Expression

25. Protein Expression PCR gene

26. Protein Expression PCR gene E. coli

27. Protein Expression PCR gene plasmid E. coli DNA extract

28. Protein Expression PCR gene plasmid

29. Protein Expression PCR gene cut plasmid plasmid

30. Protein Expression PCR gene recombinant plasmid plasmid

31. Protein Expression PCR gene recombinant plasmid plasmid E. coli transform

32. Protein Expression PCR gene E. coli E. coli growth recombinant plasmid plasmid E. coli transform

33. Protein Expression E. coli E. coli lysis

34. Protein Purification

35. Protein Purification

36. How much do proteins cost?

37. How much do proteins cost? • Gold: $450/ounce

38. How much do proteins cost? • Gold: $450/ounce • Lysozyme: $18,000/ounce

39. How much do proteins cost? • Gold: $450/ounce • Lysozyme: $18,000/ounce • HIV protease: ~$109/ounce

40. How much do proteins cost? • Gold: $450/ounce • Lysozyme: $18,000/ounce • HIV protease: ~$109/ounce • Antimatter: ~$1015/ounce

41. Protein Purification

42. How do you get the structure? • Purify the protein • Crystallize it • Record x-ray diffraction patterns • Calculate electron density • Build an atomic model

43. Protein Purification

44. Crystallize it

45. Crystallize it

46. Crystallize it

47. Crystallize it

48. Crystallize it

49. How do you get the structure? • Purify the protein • Crystallize it • Record x-ray diffraction patterns • Calculate electron density • Build an atomic model

50. Mount The Crystal