1. Structure of Oxalyl-CoA decarboxylase solved from a hemihedrally twinned crystal Supervisor Prof. Ylva Lindqvist Molecular Structural Biology Medical Biochemistry and Biophysics Karolinska Institutet Catrine Berthold, PhD student

2. Oxalate Characteristics: Highly oxidized Strong chelator of cations Toxic to most life forms

3. Oxalate Accumulation Oxalate Sources: Diet Normal cellular metabolism Mammals do not have any oxalate catabolism Related diseases: High concentration is fatal Lower concentrations: –Hyperoxaluria –Cardiac conductance disorders, cardiomyopathy –Renal failure and Ca-oxalate stones (Urolithiasis)

4. Oxalobacter formigenes Depends on oxalate as unique energy and carbon source Colonizes the gastrointestinal tract of most vertebrates Symbiotic relationship with mammals

5. Formate 1- Oxalyl-CoA Formyl-CoA Oxalate 2- Formyl-CoA transferase Oxalyl-CoA decarboxylase Oxc Frc Ricagno, S. et al. (2003) EMBO J. 22, 3210-3219.

6. Oxc crystal form 1 Space group: P4 2 2 1 2 Resolution: 4.1 Å Molecular Replacement: Program: MolRep Template: (23% ident.) Modified Acetohydroxyacid synthase CC: 0.528 Incomplete model

7. Oxc crystal form 2 Space group:P3 1 21 Resolution:1.73 Å Twin fraction: ~ 0.44

8. Data collection statistics High resolution passLow resolution pass BeamlineID23-1 ESRF Wavelength (Å)0.9757 Resolution (Å)29.24-1.73 (1.82-1.73)62.02-2.32 (2.45-2.32) Space groupP3 1 21 a, b (Å) c (Å) 124.3 151.7 No. observations1004713 (145215)272295 (10045) No. unique140751 (20434)41398 (1360) R sym (%)9.8 (49.2)6.0 (22.5) Completeness (%)99.8 (100.0)79.1 (79.1) Multiplicity (%)7.1 (7.1)6.0 (1.4) Mean {I/  (I)} 17.9 (3.8)22.4 (2.3) Wilson B factor (Å 2 )21.139.4

9. What is twinning? Non-merohedral (epitaxial) Merohedral - Hemihedral - two domains Dauter, Z. (2003) Acta Cryst. D59, 2004-2016.

10. Merohedral twinning Single untwinned crystal with two domainsHemihedrally twinned crystal Perfect  = 0.5, Partial  < 0.5  = twin fraction

11. Possible twins True point groupTwin operationhkl related to Apparent point group 32 along a,bh, -h-k, -l321 2 along a*,b*h+k, -k, -l312 2 along c-h, -k, l6 42 along a,b,a*,b*h, -k, -l422 62 along a,b,a*,b*h, -h-k, -l622 3212 along c,a*,b*-h, -k, l622 3122 along c,a,b-h, -k, l622 234 along a,b,ck, -h, l432 (If perfect twin)

12. Hemihedral twinning Intensities I obs (h 1 ) = (1 –  ) I(h 1 ) +  I(h 2 ) I obs (h 2 ) =  I(h 1 ) + (1 –  )I(h 2 ) I(h 1 ) = [ (1 –  ) I obs (h 1 ) –  I obs (h 2 )] / (1 – 2  ) I(h 2 ) = [ –  I obs (h 1 ) + (1 –  ) I obs (h 2 )] / (1 – 2  ) Detwinning when   0.5

13. Cumulative intensity distribution Z = I /  I  Howells, E.R. et al. (1950). Acta Cryst. 3, 210-214 Stanley E. (1972). J Appl. Cryst. 5, 191-194.Rees, D. C. (1980). Acta Cryst. A36, 578-581.

14. Wilson Ratio Untwinned Perfectly twinned Average over all resolution shells = 0.819

15. Space group ambiguity 622 6 321 312 3 (tetartohedral twinning) 2 along c, a, b 2 along c, a*, b* 2 along a, b, a*, b* Apparent symmetry: Possible true point symmetries:

16. Oxc Data P622P6P321P312 R merge 0.0790.0770.0600.078 Nmol/asym unit1222 Twin fraction (α)0.000.480.440.48 Yeates & Fam ‘Merohedral Crystal Twinning Server’ http://www.doe-mbi.ucla.edu/Services/Twinning/

17. Assigning space group MR statistics:P6 2 P6 4 P3 1 21P3 2 21 P3 1 12P3 2 12 R factor 55.455.359.761.050.850.7 Corr. coef.20.219.923.918.221.020.4 10 cycles of Rigid body refinement in CNS R twin 44.644.243.544.544.644.1 R free 43.444.541.942.646.647.0

18. Self rotation analysis  = 180˚ = P3 1 21 Crystallographic Twinned dataData detwinned in P3 1 21 NCS parallel to twinning axis

19. The Yeates S(H) plot Estimation of twin fraction Yeates, T.O. (1988). Acta Cryst. A44, 143-144. H = [I obs (h 1 ) – I obs (h 2 )] / [I obs (h 1 ) + I obs (h 2 )]  H  = 0.5 –   H 2  = (1 – 2  ) 2 /3 For acentric reflections S(H) = H / (1 – 2  )

20. Estimation of twin fraction Britton Plot of negative intensities Britton, D. (1972). Acta Cryst. A28, 296-297; Fisher, R.G. & Sweet, R.M. (1980). Acta Cryst. A36, 755-760

21. Local intensity statistics Padilla, J.E. & Yeates, T.O. (2003). Acta Cryst. D59, 1124-1130 Untwinned Perfect twin Twinned data Detwinned  = 0.43 Detwinned  = 0.385 L = [I obs (h 1 ) – I obs (h 2 )] / [I obs (h 1 ) + I obs (h 2 )] where h 1 and h 2 are unrelated reflections For acentric data N(|L|) = | L|

22. Estimated twin fraction  Britton plot0.43 Yeates statistics0.43 HH  H 2  Local intensity statistics 0.42 0.39

23. Refinement CNS- twin refinement protocol –Minimization –Bindividual SHELXL- refinement of twin fraction Current values:  = 0.440 R twin = 14.9 R free = 16.1

24. Structure- Tetramer Oxalyl-CoA decarboxylase (Oxc)

25. Density ADP Thiamin diphosphate 2FoFc- 1.5  FoFc- 4  Mg 2+

26. NCS promotes twinning 6.5Å shift Twin domain 1 Twin domain 2 90˚

27. Twin domains Twin domain 1 Twin domain 2 Monomer B in blue Twin axis- 180˚ along c

28. Acknowledgements Supervisors: Prof. Ylva Lindqvist & Prof. Gunter Schneider Collaborators: Prof. Nigel Richards Patricia Moussatche (Unv. of Florida) Special thank to all my co- workers in the Molecular Structural Biology group at the Karolinska Institute