shelxl: Refinement of Macromolecular Structures from Neutron Data

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1 ESS Neutron Protein Crystallography 2013 Aarhus, Denmark shelxl: Refinement of Macromolecular Structures from Neutron Data Tim Grüne University of Göttingen Dept. of Structural Chemistry Tim Grüne Shelxl for MX Neutron Data 1/26

Motivation for Neutron Macromolecular Crystallography X-ray scattering f(2θ = 0) = Z/Å Neutron scattering length b c H D C N O P Ag H D C N O P Ag Pharmaceutic application:

2 Motivation for Neutron Macromolecular Crystallography X-ray scattering f(2θ = 0) = Z/Å Neutron scattering length b c H D C N O P Ag H D C N O P Ag Pharmaceutic application: Binding interaction between drug and target usually hydrophobic Acting interaction usually electrostatic / interchange of hydrogens Tim Grüne Shelxl for MX Neutron Data 2/26

3 Benefit of neutron radiation 1. Detection of H/D atoms (enzymatic reactions) 2. Detection of metals in their reduced state (e.g. F e 2+ in rubredoxin, 4AR4 vs. 4AR3) no production of free radicals, i.e. no radiation damage (see M. Cuypers et al. Near-Atomic Resolution Neutron Crystallography on Perdeuterated Pyrococcus Furiosus Rubredoxin: Implication of Hydronium Ions and Protonation State Equilibria in Redox Changes., Angew. Chem., Int. Ed. (2013) 52, pp ) Tim Grüne Shelxl for MX Neutron Data 3/26

4 Generation of Deuterated Crystals 1. Small Molecules: Storage in D 2 O (> 3 months) only replaceable H, e.g. not methyl groups CH 3 2. Proteins: recombinant expression by E. coli with fully deuterated media (D 2 O, amino acids,... ) Tim Grüne Shelxl for MX Neutron Data 4/26

5 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 cutoff) Cambridge Crystallographic Database (Nov 2012) 624,927 entries, 1,565 neutron data Tim Grüne Shelxl for MX Neutron Data 5/26

6 Joint X ray + Neutron data Refinement 1978 A.G. Orpen, D. Pippard, and G.M. Sheldrick: Decacarbonyl-µ-hydrido-µ-vinyl-triangulo-triosmium (Acta Cryst. B34, ) modified version of SHELX76, source code no longer available 1982 Wlodawer, Hendrickson, PROLSQ, Acta Cryst. A P. D. Adams et al., PHENIX.REFINE, Acta Cryst. D65, Stabilisation of refinement by X ray data Tim Grüne Shelxl for MX Neutron Data 6/26

7 Why Neutron Refinement Alone ESS Neutron Protein Crystallography 2013 Main point of neutron structure solution: protonation states of carboxyl groups, histidine,... X-rays interact with electrons, i.e. hydrogen bonds from X-ray data appear shorter (0.9Å) than internucleic distances (1.1Å) positions of hydrogen in water and OH groups cannot be inferred from X-ray data While weak, hydrogen contribution is taken into account when calculating X-ray structure factors X-ray and neutron data are different This work: Feasibility study of refinement against neutron data alone Data: Rubredoxin (provided by Dr. Flora Meilleur) Program: SHELXL-2013 Tim Grüne Shelxl for MX Neutron Data 7/26

8 Neutron Refinement with Shelxl-97 Previously (shelxl-97): Scattering bond length defined through SFAC card SFAC C SFAC H SFAC N CHIV-restraint (chiral volume) does not work for C α bound to 4 atoms: ** Bad CHIV: CA_1043 bonds to DA_1043 N_1043 CB_1043 C_ ignored ** important restraints for protein structures missing Tim Grüne Shelxl for MX Neutron Data 8/26

9 Shelxl-2012: NEUT card 1. Removes special treatment of hydrogen atoms 2. Short ( normal ) SFAC syntax can now be used: SFAC C H N Convenient consequence: compatibility with COOT (P. Emsley) 3. CHIV CA works Tim Grüne Shelxl for MX Neutron Data 9/26

The Rubredoxin Neutron Data A. S. Gardberg et al., Acta Cryst. (2010) D66, 558 567 PDB codes 3kyu, 3kyv, 3kyw, 3kyx, and 3kyy Neutron data for 3kyx kindly provided by Dr.

10 The Rubredoxin Neutron Data A. S. Gardberg et al., Acta Cryst. (2010) D66, PDB codes 3kyu, 3kyv, 3kyw, 3kyx, and 3kyy Neutron data for 3kyx kindly provided by Dr. Flora Meilleur (SNS Oak Ridge, USA) 3kyx: joint neutron + X-ray refinement, T=295 K, 1.6 Å, R/R work = 16.9%/19.7% P , a = b = c = Tim Grüne Shelxl for MX Neutron Data 10/26

11 Rubredoxin Neutron Data from LADI-III at ILL Tim Grüne Shelxl for MX Neutron Data 11/26

12 Data and Parameters for Rubredoxin, T = 295K λ Å d Å ( Å) # reflections unique 4781 (237) Completeness 72.1 % (30.3%) 817 atoms + 1Fe 2+ or 3+ (C H 396 N 62 O 87 S 5 ) (excluding solvent molecules) Tim Grüne Shelxl for MX Neutron Data 12/26

Conformation of ILE 1011 not present in 3kyx Fragment of C-terminal GLU 1052 not present in

13 ESS Neutron Protein Crystallography 2013 Refinement Results R/Rfree = 22.5%/29.5% (4256 reflections for 2923 parameters) No disagreeable restraints Confirmative: Dual Conformation of ILE 1011 not present in 3kyx Fragment of C-terminal GLU 1052 not present in 3kyx Conservative placement of water molecules: 11 D2O vs. 28 in 3KYX Tim Grüne Shelxl for MX Neutron Data 13/26

14 Restraints and Constraints Data to parameter ratio very low (< 1.5 without restraints) Neutron data: many more parameters than X-ray because of hydrogen atoms Refinement must be stabilised through constraints & restraints Restraints mathematically like data, but cannot make up for lack of completeness Constraints reduce number of parameters, i.e. real improvement of data:parameters Tim Grüne Shelxl for MX Neutron Data 14/26

15 Standard Protein Restraints & Constraints SHELXPRO: Standard restraints for macro molecules (Engh & Huber Parameters, Acta Cryst. (1991), A47, ) derived from CCSD: DFIX, DANG, CHIV, FLAT Treatment of hydrogens in X-ray crystallography: Riding hydrogen model = calculated H-positions relative to bonded atom (HFIX/ AFIX in shelxl) = no extra parameters for X-ray data Tim Grüne Shelxl for MX Neutron Data 15/26

16 New Restraints for D/H F.H. Allen, I.J. Bruno: Bond lengths in organic and metal-organic compounds revisited: X-H bond lengths from neutron diffraction data, Acta Cryst (2010), B66, D Only very few samples, e.g. searching for C N 7 C results in # 15 samples from CCSD Guesstimate distances (H-X distance for X-ray data in shelxl set 40 years ago... ) Default effective X-H distances for T = 20.0 C AFIX m = [N] 3[N] 15[B] 8[O] 9 9[N] 16 d(x-h) = Tim Grüne Shelxl for MX Neutron Data 16/26

5 1.096 CB HFIX_ASN 93-1.5 1.02 ND2 Generation of Angular Distances restraints(dang): measured from resulting.

17 Hydrogenation with shelxl Coordinate for non-h/ non-d usually known from X-ray experiment Addition of hydrogens with preset distance in shelxl with "HFIX" commands HFIX_ASN N HFIX_ASN CA HFIX_ASN CB HFIX_ASN ND2 Generation of Angular Distances restraints(dang): measured from resulting.res-file after 0 cycles of refinement Tim Grüne Shelxl for MX Neutron Data 17/26

18 More chemistry: CHIV vs. FLAT Cζ Cζ CHIV CHIV vs. FLAT CHIV CZ : more flexibility for D atom to move out of aromatic ring CHIV N available for neutron data Tim Grüne Shelxl for MX Neutron Data 18/26

19 Constraining U iso (D/H) SHELXL syntax: U iso = 5 q 0.5: Isotropic ADP relative to previous normal atom U iso (D) = q U iso (X) avoid 1 parameter per D automatic with e.g. HFIX_ASN N Heuristic values by bond type: N D 2/3-1.7 C a D a -1.3 N D -1.3 C D O D -1.7 C D R/R free = 22.9%/30.8% 23.6%/30.1% = Closure of gap between R and R free Tim Grüne Shelxl for MX Neutron Data 19/26

20 Deuterium Saturation Motivation: Contamination of 2 H with 1 H shelxl: Occupancy refinement for groups of atoms by free variables type exchange rate FVAR N D & O D easily exchanged 2 C α D α medium exchange 3 C D n slow to no exchange 4 Starting values for all three FVAR s: 0.9 Tim Grüne Shelxl for MX Neutron Data 20/26

21 Deuterium Saturation 1. Non-physical results for carbon bound D type refined FVAR N D n & O D C α D α non-sense C D n non-sense 2. Only Nitrogen and Oxygen bound D: FVAR=89.3% 3. Calculation of D saturation p: f b c (D) = p b c (D) + (1 p) b c (H) fm = p 6.67fm (1 p) 3.74fm p = 93% 4. Easier than dual conformation with PARTs for D and H Tim Grüne Shelxl for MX Neutron Data 21/26

22 Data:Parameter: Replacing R free with 50-fold cross-validation Macromolecular Refinement without cross validation nearly impossible (over-fitting) A. Brunger (Nature, 1992 (355), p.472): reflections necessary 50-fold cross validation with 50 flagged reflections (conventional: 22.5%/29.5%): R 1 : 22.9% ± 0.06% (R free ) : 29.6% ± 3.73% Benefit: 4731 vs reflections, ratio 1.62 vs Tim Grüne Shelxl for MX Neutron Data 22/26

Validation of Restraints ESS Neutron Protein Crystallography 2013 PDB ID 4AR4: Neutron data-only Rubredoxin data set (1.4Å resolution) by M. Cuypers et al.

23 Validation of Restraints ESS Neutron Protein Crystallography 2013 PDB ID 4AR4: Neutron data-only Rubredoxin data set (1.4Å resolution) by M. Cuypers et al. Near-Atomic Resolution Neutron Crystallography on Perdeuterated Pyrococcus Furiosus Rubredoxin: Implication of Hydronium Ions and Protonation State Equilibria in Redox Changes., Angew. Chem., Int. Ed. (2013) 52, pp data available at PDB contain I+ and I, not only F rubredoxin ins-file refined against r4ar4sf.ent geometry checked with Tim Grüne Shelxl for MX Neutron Data 23/26

24 Validation of Restraints PDB-ID 4AR4 4AR4 with shelxl restraints, no model building 4AR4 with shelxl restraints, after 1 st round of model building Tim Grüne Shelxl for MX Neutron Data 24/26

25 Conclusions MX refinement of neutron data alone feasible SHELXL provides powerful constraints (Uiso = -1.7) Occupancy refinement for D gives reasonable result TODO: DNA restraints (PDB ID 1WQZ and 3QBA) Tim Grüne Shelxl for MX Neutron Data 25/26

26 Acknowledgment Dr Flora Meilleur, Spallation Neutron Source, Oak Ridge, USA: Neutron data sets for Rubredoxin and Xylose Isomerase Prof George M. Sheldrick, University of Göttingen for discussion, advice and for SHELXL-2013 Thomas Niklas (model building of 4AR4) Tim Grüne Shelxl for MX Neutron Data 26/26

4. Constraints and Hydrogen Atoms

4. Constraints and Hydrogen Atoms 4. Constraints and ydrogen Atoms 4.1 Constraints versus restraints In crystal structure refinement, there is an important distinction between a constraint and a restraint. A constraint is an exact mathematical