Crystallization of macromolecules

1. This presentation will probably involve audience discussion, which will create action items. Use PowerPoint to keep track of these action items during your presentation • In Slide Show, click on the right mouse button • Select “Meeting Minder” • Select the “Action Items” tab • Type in action items as they come up • Click OK to dismiss this box This will automatically create an Action Item slide at the end of your presentation with your points entered. Crystallization of macromolecules Biology 555 Andrew J. Howard Crystallization and Phasing

2. krystallos? krustallos? krustallos Crystallization and Phasing

3. Who What When Where Why How Whither Whence (Wherefore) Journalist’s Criteria Crystallization and Phasing

4. Who makes macromolecular crystals? • Macromolecular crystals are almost always produced artificially, i.e., by human action • So “who” is “scientists” Crystallization and Phasing

5. What are macromolecular crystals? • Crystals are translationally ordered arrays of molecules • Macromolecular crystals are held together by relatively weak ionic intermolecular forces • Solvent content generally above 40% Crystallization and Phasing

6. When have we made them? • Story goes back into the mid-19th century • Systematic search for crystallization conditions dates from the 1950’s • Screening kits: concept 1980’s, commercialization in 1990’s • Robotics late 1990’s • Nanoscale techniques early 21st century • Growth of huge numbers of nanocrystals: 2012-present Crystallization and Phasing

7. Crystallization • Very old (reproduction, genetic duplication) • Empirical (trial & error -- ‘Screens’) • ‘Art’ vs ‘Science’ Crystallization and Phasing

8. Short History • 1853 HemoglobinLehman, CG. Lehrbuch der physiologischeChemie. Leipzig • 1926 Urease Sumner, JB. J Biol Chem. 69: 435 • 1930 Pepsin & other proteolytic enzymes Northrop, JH, Kunitz, M, Herriot RM. Crystalline Enzymes. Columbia University Press, NY (review) • 1934 Pepsin DiffractionBernal JD & Crowfoot, D. Nature, 133:794 • 1935 Tobacco Mosaic VirusStanley, WM. Science. 81: 644 1946 Nobel (Chemistry) Crystallization and Phasing

9. 1946:Sumner Nobel prize 1958: Myoglobin structure 1959: Hemoglobin structure 1962: Perutz/Kendrew Nobel 1979: Carter & Carter paper 1985: first microgravity experiments 1990’s: commercial screening kits Late 1990’s: viable commercial crystallization robots Early 2000’s: microfluidics ~2012: huge quantities of nanocrystals Short history (concluded) Crystallization and Phasing

10. Where do we grow them? • Under mild laboratory conditions • Contrast to inorganic small molecules, which are often grown from a melt • Even small organic crystals often exploit temperature dependence • Proteins usually avoid these techniques… Crystallization and Phasing

11. Growing [Protein] Crystals • Inorganic - cooling a hot saturated substance • Polar organic - same or ppt from aqueous using organic solvents • Proteins - Yeoww!! (denature) • Dissolved in buffer + ppt [low] • controlled evaporation [higher] Crystallization and Phasing

12. Why do we grow them? • Because we want to know the macromolecule’s structure! • Fundamental postulate:The structure of a macromolecule in a crystal differs only slightly, and then only on the surface, from that of its soluble or membrane-associated (biologically active) form • N.b.: do we care about the surface? depends on the protein. • With enzymes, no • with antibodies, often • Everything else ?? Crystallization and Phasing

13. Do we believe this? • Short answer: yes • Skepticism was rampant through the 1970’s and has only gradually diminished • Various experimental demonstrations Crystallization and Phasing

14. Evidence thatr(xtal) = r(solution) • Enzymatic activity of crystals (1970s’s) • Similarity of multiple crystal forms(why does this help? If two structures of the same protein, having completely different crystal contacts, look the same, then they’re probably both equivalent to the solution structure) • Comparisons to NMR structures • Consistency with other biophysical techniques Crystallization and Phasing

15. When is the postulate wrong? • Some external loopsheld in wrong positions(Interleukin-8) • Much more common: crystal structure shows us one conformer; other conformers, and the transitions among them, are relevant Crystallization and Phasing

16. How to make 3-D crystals • In general it involves creation of three-dimensional order • In practice with macromolecules that means creating conditions in which intermolecular forces can be exerted in the same way on each molecule • These intermolecular forces are • Polar • Often water-mediated • Weak Crystallization and Phasing

17. How do we grow macromolecular crystals? • Short answer: we gradually decrease the solubility of the protein in a way that produces ordered (crystalline) precipitation rather than disordered (amorphous) precipitation • Recognize the stages in crystallization Crystallization and Phasing

18. Stages of crystallization • Nucleation • Governed by short-range intermolecular interactions • We want a few stable nuclei, not a lot! • Growth • Adding one molecule at a time to the nucleus • Incorrect additions lead to instability and… • Cessation of growth Crystallization and Phasing

19. Protein Preparation • Know your protein • Cysteines • Substrates / Ligands • Proteolytic sensitivity • Metal binding • pH & Temp for stability / activity • Post Translational Modification Crystallization and Phasing

20. Protein Preparation • Purify from natural sources • Create an expression construct • Add Tags to aid purification • 6-His, Biotin-Strept., Calmodulin Bind. Peptide, GST, Maltose Bind. Protein • Expression systems • E.coli - no post translational modifs • Yeast – eukaryotic:may be better for secreted proteins • Baculovirus-Insect & Mammalian cells Crystallization and Phasing

21. Protein Preparation • Purification Strategy • Optimize Protein Expression • Preparation of soluble cell-free extract • AMS/PEG fractionation • Affinity Chromatography • Ion Exchange Chromatography • Size Exclusion Chromatography • Homogeneity Analysis (SDS-PAGE, MS, DLS) Crystallization and Phasing

22. Protein vs Salt Keep Protein Crystals Hydrated in “Mother Liquor” Crystallization and Phasing

23. Solubility Curve Zone 3 – Precipitation. Generally amorphous Zone 2- Nucleation crystals grow Zone 1 - Metastable rare nucleation sustains growth (seed) Below S - no ppt Crystallization and Phasing

24. Does it always work this way? • No. Some proteins are more soluble in high salt than low. • Same general principles apply as long as we understand the dynamics Crystallization and Phasing

25. What does aggregation do? • In a sense, a crystal is an aggregate… • The formation of oligomeric, randomly oriented aggregates is not conducive to crystallization • Light-scattering experiments can detect aggregation Crystallization and Phasing

26. Second virial coefficient • Characterizes two-body interactions between (protein) molecules in dilute solution Crystallization and Phasing

27. What do we do with that? • It can be measured through static light-scattering and SANS measurements • Good correlation with nucleation conditions, at least with favorable proteins Crystallization and Phasing

28. Crystallization Methods • Batch • Dialysis • Vapor Diffusion Crystallization and Phasing

29. Dialysis Crystallization and Phasing

30. Double Dialysis Crystallization and Phasing

31. Vapor Diffusion Crystallization and Phasing

32. Vapor Diffusion Variants Crystallization and Phasing

33. Factors affecting Crystallization • Physical:Temp, pressure, surface, viscosity, vibration • Chemical:pH, precipitant, ionic strength, metals • Biochemical:purity, ligands, post-TL, proteolysis • Engineering:solubility, fusion proteins, heavy atom sites Crystallization and Phasing

34. Don’t be deceived!http://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial1.html Beautiful - No diffraction Ugly - 1.6 Ångstroms ! Moral : Its the diffraction that counts Crystallization and Phasing

35. Precipitateshttp://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial2.htmlPrecipitateshttp://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial2.html • Good - nonamorphous, birefringent, redissolves • Bad - skin, does not redissolve, characteristic brownish tinge Crystallization and Phasing

36. http://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial2.htmlhttp://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial2.html Crystallization and Phasing

37. Whence came we? • It used to be really hard: • Inadequate quantity • Inadequate purity • Unsystematic approaches • Macro quantities required • Motivation to improve crystallization approaches came as the field matured Crystallization and Phasing

38. Whither? • High-throughput • Better protein purity • Higher quantities when required • Approaches that don’t require large quantities have appeared • More systematic approaches • Automation at every stage, including visualization Crystallization and Phasing

39. Automated crystallization • Sample loading, distribution • visualization, decision-making Crystallization and Phasing

40. Can we get away with not knowing our protein? • Often, yes:cf. Structural genomics projects • Ugly cases (e.g. transmembrane):we still argue that the more you know, the more likely you are to get good crystals Crystallization and Phasing

41. Advances in the 2000’s • The development of microfluidics allows the scale of protein crystallization experiments to go down to the sub-microliter level • Scale: 1 nL drop => 0.1 nL xtal = 10-10L = 10-13m3 = (2*10-5m)3 = (20 µm)3 • Below ~20nL (=> 5µm xtals) the crystals may actually not be big enough to use for synchrotron experiments, but they can still serve as a guide for scaleup to ~50 nL Crystallization and Phasing

42. The role of free electron lasers • First FELs built mid-2010’s: Stanford, then Pohang, Hamburg • Xtals are flowed past the Xray beam, which is so intense that each crystal is completely destroyed by the shot • Most of the crystals never see beam • So you need lots of crystals! • First experiments: 105 ~1 µm crystals! • Better now because they’re flowing through toothpaste rather than water: only ~103 xtals Crystallization and Phasing

43. References • www.hamptonresearch.com • www.emeraldbiostructures.com • Protein Crystallization (ed. T. Bergfors)http://xray.bmc.uu.se/~terese/ • Crystallization of Nucleic Acids and Proteins(ed. A. Ducruix & R. Giege) • http://www.hwi.buffalo.edu/High_Through/High_Through.html • International Tables for Crystallography. Vol. F • Part 3 : Techniques of Molecular Biology(S. Hughes & A. Stock) • Part 4 : Crystallization (R. Giege et al.) Crystallization and Phasing