Neutron Diffraction and
Scattering in Biology
Penghui Lin
Department of Physics
Oklahoma State University
10/4/2013
Neutron Scattering
• Neutron Reflection (Neutron Reflectometry)
• Small Angle Neutron Scattering
• Neutron diffraction (Neutron Crystallography)
• Spectroscopy and Imaging
Structure determination
• X-ray diffraction----spatial distribution of
electrons
• Electron diffraction----Coulomb forces
• Neutron diffraction---strong nuclear forces
X-ray
• NMR
• IR
NMR
Electron Microscopy
Hybrid
Other
X-ray vs Neutron
X-ray v.s. Neutron Crystallography
Crystal -> Diffraction pattern -> Electron density -> Model
Crystal -> Diffraction pattern -> Nuclear density -> Model
X-ray v.s. Neutron
Information from NC
• Equivalence: neutron scattering not strongly depends
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
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
Neutron diffraction in structural studies
•
•
•
•
Location of Hydrogen atoms
Solvent Structure
Hydrogen exchange
Low resolution studies
H
D
%
bc
bi
99.985 -3.741 25.27
0.015 6.671 4.04
sc
1.758
5.592
si
80.27
2.051
ss
82.03
7.643
sa
0.3326
0.000519
Bound atom scattering length
Neutron sees
Fissure Reactor
Chained 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
Neutron source
Neutron source
Combined with Fourier Transform
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)
Solution
• 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
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
New neutron sources
Applications
EXAMPLE I
D-XYLOSE ISOMERASE (XI)
XI: Xylose Isomerase
Mechanism of Aldo to Keto
Environment
OD- in XI-xylulose
D2O in native XI
M1: structural metal
M2: catalytic metal
Kovalevsky 2008 Biochemistry
Active site of XI-xylulose
Doubly protonated
singly protonated
Kovalevsky 2008 Biochemistry
Kovalevsky 2008 Biochemistry
Applications
EXAMPLE II
CRYO COOL CONCANAVALIN A
Concanavalin A
Saccharide-binding protein
Legume lectin family
Extensice β-sheet arrangement
Two metal binding sites
PDB: 3CNA
Waters in the saccharide-binding site
293K
Habash 2000 Acta Crystallogr D Biol Crystallogr.
15K
Blakeley 2004 PNAS
H-bond network
Blakeley 2004 PNAS
Water comparison
Compare to room temperature NC
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
Compare to low temperature (100K) XC
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%)
Blakeley 2004 PNAS
Conserved water molecules
W1
W6
W75
Neutron 15K
Neutron 293K
X-ray
110K
Only 22 water molecules are conserved in positions
Blakeley 2004 PNAS
Applications
EXAMPLE III
SANS IN LIPID UNIFORMITY
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
Fluorescence microscopy of GUVs
ρ ≡ χDOPC/(χDLPC+χDOPC)
FRET and SANS results
Small Angle Neutron Scattering
Förster Resonance Energy Transfer
Conclusion
• A complementary technique to others
• Sensitive to light atoms, especially hydrogen
• Can be applied to various materials
References
1. Heberle FA, et al. (2013) Hybrid and Nonhybrid Lipids Exert Common Effects on Membrane Raft Size and Morphology. Journal of the
American Chemical Society.
2. 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.
3. Stuhrmann HB (2004) Unique aspects of neutron scattering for the study of biological systems. Rep Prog Phys 67(7):1073-1115.
4. 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.
5. Holt SA, et al. (2009) An ion-channel-containing model membrane: structural determination by magnetic contrast neutron
reflectometry. Soft Matter 5(13):2576-2586.
6. Blakeley MP, Langan P, Niimura N, & Podjarny A (2008) Neutron crystallography: opportunities, challenges, and limitations. Curr
Opin Struc Biol 18(5):593-600.
7. 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.
8. Collyer CA & Blow DM (1990) Observations of Reaction Intermediates and the Mechanism of Aldose-Ketose Interconversion by DXylose Isomerase. Proceedings of the National Academy of Sciences of the United States of America 87(4):1362-1366.
9. 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.
10. 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.
11. Lakey JH (2009) Neutrons for biologists: a beginner's guide, or why you should consider using neutrons. J R Soc Interface 6:S567S573.
12. Kovalevsky AY, et al. (2008) Hydrogen location in stages of an enzyme-catalyzed reaction: Time-of-flight neutron structure of Dxylose isomerase with bound D-xylulose. Biochemistry-Us 47(29):7595-7597.
Thanks