1. Neutron diffraction & scattering
Andy Howard Biology 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 = m
Therefore we need neutrons with momentum p = h/l = 6.626*10-34 J-sec/(10-10m) p = 6.626*10-24 kg m s-1
If nonrelativistic then v = p/m v = (6.626*10-24 / 1.675*10-27) m s-1 v = 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 = *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 bi sc si ss sa H D

16. Amino acid scattering lengths

17. Nucleic acid bases

18. Water & lipids

19. Neutron sees

20. Fission Reactor Spallation source 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 Solutions
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 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: In spallation:
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. Example I D-Xylose Isomerase (XI)
PDB 3KCO Joint neutron and X-ray refinement
Applications
Example I D-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. Example II: concanavalin A
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
Neutron 15K
Neutron 293K
X-ray K
Only 22 water molecules are conserved in positions
Blakeley 2004 PNAS

40. Example iii SANs in lipid uniformity
Applications
Example iii SANs 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):
Stuhrmann HB (2004) Unique aspects of neutron scattering for the study of biological systems. Rep Prog Phys 67(7):
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:
Holt SA, et al. (2009) An ion-channel-containing model membrane: structural determination by magnetic contrast neutron reflectometry. Soft Matter 5(13):
Blakeley MP, Langan P, Niimura N, & Podjarny A (2008) Neutron crystallography: opportunities, challenges, and limitations. Curr Opin Struc Biol 18(5):
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):
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):
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):
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):
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):