Neutron diffraction & scattering

1. Neutron diffraction & scattering Andy HowardBiology 555, 26 September 2016 Based on lecture prepared by Penghui Lin, Oklahoma State University

2. Agenda • Neutrons and waves • Comparing Neutrons with X-rays • Neutron sources • Neutron crystallography • Examples • Solution scattering using neutrons

3. Neutrons as tools in biological research • Neutron Reflection (Neutron Reflectometry) • Small Angle Neutron Scattering • Neutron diffraction (Neutron Crystallography) • Spectroscopy and Imaging • Magnetic scattering from nuclei

4. How does this work at all? • DeBroglie recognized that the wave-particle duality for light could also apply to particles • Davisson-Germer experiment (electrons traveling through a 2-slit assembly) showed that electrons could interfere • Neutrons should have wavelike behavior with a wavelength l = h/p

5. Length and velocity scales • We want to look at structures at atomic-bond resolution level, i.e. around 1Å = 10-10 m • Therefore we need neutrons with momentump = h/l = 6.626*10-34 J-sec/(10-10m)p = 6.626*10-24 kg m s-1 • If nonrelativistic then v = p/mv = (6.626*10-24 / 1.675*10-27)m s-1v = 3960 ms-1 (nonrelativistic)

6. Kinetic energy scale • Two equivalent ways to do this: • KE = ½ mv2= 0.5*1.675*10-27 kg* (3960 m s-1)2= 1.313 *10-20 J = 819 eV • KE = p2/(2m) =(6.626*10-24)2/(2*1.675*10-27) J = 1.313*10-20 J = 819 eV • Slower than relativistic but faster than thermal neutrons (3/2)kT

7. Structure determination • X-ray diffraction----spatial distribution of electrons • Electron diffraction----Coulomb forces • Neutron diffraction---strong nuclear forces • NMR • IR

8. X-ray versus Neutrons

9. X-ray vs. Neutron Crystallography Crystal -> Diffraction pattern -> Electron density -> Model Crystal -> Diffraction pattern -> Nuclear density -> Model

10. X-ray vs. Neutron

11. Information from neutron crystallography • Equivalence: neutron scattering not strongly dependent on Z (especially for hydrogen detection which X-ray or electron diffraction can not see) • Clearly distinguish between neighboring atoms. (For biology, particularly N, C and O) • Contrast between H and D • Locate the solvent orientation around protein • Thermal motions of hydrogen containing groups • Weak interaction with materials, deep penetration and non-destructive

12. Crucial Hydrogen • Dominance of H2O molecules in living cells • Hydrogen bonds provide stability and versatility for biological macromolecules • Proton transfer and exchange is critical in many reactions • Hydration and protonation states are important

13. When do we need H’s? • Water hydrogens • Serine, threonine, tyrosine OH’s • Cysteine SH’s • Some nucleotides, but not many • Phosphate protons, esp. at low pH • Amine hydrogens in nucleic acid bases, lysine • Some ligand hydrogens

14. When don’t we need them? • Any carbon or nitrogen that is bonded to two other atoms will have its proton positions fully determined by the coordinates of the neighboring atoms! • Methyl hydrogens in principle matter but they’re usually disordered

15. Neutron diffraction in structural studies • Location of Hydrogen atoms • Solvent Structure • Hydrogen exchange • Low resolution studies % bc biscsisssa H 99.985 -3.741 25.27 1.758 80.27 82.03 0.3326 D 0.015 6.671 4.04 5.592 2.051 7.643 0.000519

16. Amino acid scattering lengths

17. Nucleic acid bases

18. Water & lipids

19. Neutron sees

20. Fission Reactor Chain reactions Continuous flow 1 neutron per fission 180 MeV neutron 1015/cm2/s Spallation source No chain reaction Pulsed 40 neutrons per proton 30 MeV neutron 1016/cm2/s

21. Neutron source

22. Neutron source Combined with Fourier Transform

23. Main problem • Low flux of neutron beams • Structures are large while scatterings are weak, so large single crystals are required, 1 mm3 is the limit due to the reasonable data collection time of 10-14 days per data set • Hydrogen produces a high level background (80 barn scattering factor) Solutions • Broad bandpass: maximize the neutron flux and the reflections on the detector • Cylindrical neutron image plate:LADI at ILL has a solid angle >2π • Isotope substitution of D to H

24. Developments • In reactors: • Neutron image plates • Quasi-Laue methods • In spallation: • Time of flight Laue method • Electronic detectors • New facilities and methods for sample perdeuteration and crystallization • New approaches and computational tools for structure determination

25. New neutron sources

26. PDB 3KCOJoint neutron and X-ray refinement Applications Example ID-Xylose Isomerase (XI)

27. XI: Xylose Isomerase

28. Mechanism of Aldo to Keto

29. Environment D2O in native XI OD- in XI-xylulose M1: structural metal M2: catalytic metal Kovalevsky 2008 Biochemistry

30. Active site of XI-xylulose Doubly protonated singly protonated Kovalevsky 2008 Biochemistry

31. Active-site residues in XI Kovalevsky 2008 Biochemistry

32. PDB 2YZ4 Applications Example II:concanavalin A

33. Concanavalin A Saccharide-binding protein Legume lectin family Extensive β-sheet arrangement Two metal binding sites PDB: 3CNA

34. Waters in the saccharide-binding site 293K 15K Blakeley 2004 PNAS Habash 2000 Acta Crystallogr D Biol Crystallogr.

35. H-bond network Blakeley 2004 PNAS

36. Water comparison Compare to room temperature NC Compare to low temperature (100K) XC Blakeley 2004 PNAS

37. Water structure • 15K • 227 water sites are identified with 19.2 Å2 B factor • 167 are D2O with 17.6 Å2 B factor • 60 are OD- or oxygen atoms with 32.2 Å2 B factor • 293K • 148 water sites are identified with 43 Å2 B factor • 88 are D2O with 37.8 Å2 B factor • 60 are OD- or oxygen atoms with 50.6 Å2 B factor

38. Comparing the water structures • Among the 16 buried waters, 9 matched the positions in the X-ray structure (56.3%) • Among the 211 surface waters, 35 matched the positions in the X-ray structure (16.6%)

39. Conserved water molecules W6 W1 W75 Neutron 15K Neutron 293K X-ray 110K Only 22 water molecules are conserved in positions Blakeley 2004 PNAS

40. Applications Example iiiSANs in lipid uniformity

41. Lipid raft Proposal: Hybrid lipids align in a preferred orientation at the boundary of ordered and disordered phases, lowering the interfacial energy and reducing domain size

42. Three-component lipid systems

43. Fluorescence microscopy of Giant Unilamellar Vesicles ρ ≡ χDOPC/(χDLPC+χDOPC)

44. FRET and SANS results Small Angle Neutron Scattering Förster Resonance Energy Transfer

45. Conclusion • Neutron scattering:A complementary technique to others • Sensitive to light atoms, especially hydrogen • Can be applied to various materials

46. References • Heberle FA, et al. (2013) Hybrid and Nonhybrid Lipids Exert Common Effects on Membrane Raft Size and Morphology. Journal of the American Chemical Society. • Comoletti D, et al. (2007) Synaptic arrangement of the neuroligin/beta-neurexin complex revealed by X-ray and neutron scattering. Structure 15(6):693-705. • Stuhrmann HB (2004) Unique aspects of neutron scattering for the study of biological systems. Rep Prog Phys 67(7):1073-1115. • Habash J, et al. (2000) Direct determination of the positions of the deuterium atoms of the bound water in concanavalin A by neutron Laue crystallography. Acta Crystallogr D 56:541-550. • Holt SA, et al. (2009) An ion-channel-containing model membrane: structural determination by magnetic contrast neutron reflectometry. Soft Matter 5(13):2576-2586. • Blakeley MP, Langan P, Niimura N, & Podjarny A (2008) Neutron crystallography: opportunities, challenges, and limitations. Curr Opin Struc Biol 18(5):593-600. • Niimura N, Chatake T, Ostermann A, Kurihara K, & Tanaka I (2003) High resolution neutron protein crystallography. Hydrogen and hydration in proteins. Z Kristallogr 218(2):96-107. • Collyer CA & Blow DM (1990) Observations of Reaction Intermediates and the Mechanism of Aldose-Ketose Interconversion by D-Xylose Isomerase. Proceedings of the National Academy of Sciences of the United States of America 87(4):1362-1366. • Blakeley MP, Kalb AJ, Helliwell JR, & Myles DAA (2004) The 15-K neutron structure of saccharide-free concanavalin A. Proceedings of the National Academy of Sciences of the United States of America 101(47):16405-16410. • Blakeley MP, et al. (2008) Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase. Proceedings of the National Academy of Sciences of the United States of America 105(6):1844-1848. • Lakey JH (2009) Neutrons for biologists: a beginner's guide, or why you should consider using neutrons. J R Soc Interface 6:S567-S573. • Kovalevsky AY, et al. (2008) Hydrogen location in stages of an enzyme-catalyzed reaction: Time-of-flight neutron structure of D-xylose isomerase with bound D-xylulose. Biochemistry-Us 47(29):7595-7597.