ESS Neutron Protein Crystallography 2013 ESS Neutron Protein Crystallography 2013 Aarhus, Denmark : Refinement of Macromolecular Structures from Neutron Data Tim Grüne University of Göttingen Dept. of Structural Chemistry http://shelx.uni-ac.gwdg.de [email protected]Tim Grüne Shelxl for MX Neutron Data 1/26
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ESS Neutron Protein Crystallography 2013
ESS Neutron Protein Crystallography 2013Aarhus, Denmark
shelxl: Refinement of Macromolecular Structures from Neutron Data
– Binding interaction between drug and target usually hydrophobic
– “Acting” interaction usually electrostatic / interchange of hydrogens
Tim Grüne Shelxl for MX Neutron Data 2/26
ESS Neutron Protein Crystallography 2013
Benefit of neutron radiation
1. Detection of H/D atoms (enzymatic reactions)
2. Detection of metals in their reduced state (e.g. Fe2+ in rubredoxin, 4AR4 vs. 4AR3) — no production offree radicals, i.e. no radiation damage
(see M. Cuypers et al. “Near-Atomic Resolution Neutron Crystallography on Perdeuterated PyrococcusFuriosus Rubredoxin: Implication of Hydronium Ions and Protonation State Equilibria in Redox Changes.”,Angew. Chem., Int. Ed. (2013) 52, pp. 1022–1025)
Tim Grüne Shelxl for MX Neutron Data 3/26
ESS Neutron Protein Crystallography 2013
Generation of Deuterated Crystals
1. Small Molecules: Storage in D2O (> 3 months)
⇒ only replaceable H, e.g. not methyl groups CH3
2. Proteins: recombinant expression by E. coli with fully deuterated media (D2O, amino acids, . . . )
Tim Grüne Shelxl for MX Neutron Data 4/26
ESS Neutron Protein Crystallography 2013
Problems of MX Neutron Measurements
• incomplete data, even from Laue data collection
• sample size
• costs for deuteration
Protein Data Bank (Nov 2012) 86,487 structures, 63 by neutron diffraction (29 unique by 90 % sequence cut-off)
Cambridge Crystallographic Database (Nov 2012) 624,927 entries, 1,565 neutron data
Tim Grüne Shelxl for MX Neutron Data 5/26
ESS Neutron Protein Crystallography 2013
Joint X–ray + Neutron data Refinement
1978 A.G. Orpen, D. Pippard, and G.M. Sheldrick: Decacarbonyl-µ-hydrido-µ-vinyl-triangulo-triosmium (ActaCryst. B34, 2466–272) modified version of SHELX76, source code no longer available
4. Easier than “dual conformation” with PARTs for D and H
Tim Grüne Shelxl for MX Neutron Data 21/26
ESS Neutron Protein Crystallography 2013
Data:Parameter: Replacing Rfree with 50-fold cross-validation
• Macromolecular Refinement without cross validation nearly impossible (over-fitting)
• A. Brunger (Nature, 1992 (355), p.472): 500–1000 reflections necessary
• 50-fold cross validation with 50 flagged reflections (conventional: 22.5%/29.5%):
R1 : 22.9%± 0.06% (Rfree) : 29.6%± 3.73%
• Benefit: 4731 vs. 4256 reflections, ratio 1.62 vs. 1.46
Tim Grüne Shelxl for MX Neutron Data 22/26
ESS Neutron Protein Crystallography 2013
Validation of Restraints
• PDB ID 4AR4: Neutron data-only Rubredoxin data set (1.4Å resolution) by M. Cuypers et al. “Near-AtomicResolution Neutron Crystallography on Perdeuterated Pyrococcus Furiosus Rubredoxin: Implication of Hy-dronium Ions and Protonation State Equilibria in Redox Changes.”, Angew. Chem., Int. Ed. (2013) 52, pp.1022–1025
• data available at PDB contain I+ and I−, not only F
• rubredoxin ins-file refined against r4ar4sf.ent
• geometry checked with http://molprobity.biochem.duke.edu/