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Structure of Oxalyl-CoA decarboxylase solved from a hemihedrally twinned crystal

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

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Structure of Oxalyl-CoA decarboxylase solved from a hemihedrally twinned crystal

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

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

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

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

  5. Ricagno, S. et al. (2003) EMBO J. 22, 3210-3219. Oxalate2- Formate1- Frc Formyl-CoA transferase Formyl-CoA Oxalyl-CoA Oxalyl-CoA decarboxylase Oxc

  6. Oxc crystal form 1 Space group:P42212 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: P3121 Resolution: 1.73 Å Twin fraction:~ 0.44

  8. Data collection statistics

  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 domains Hemihedrally twinned crystal • = twin fraction Perfect  = 0.5, Partial  < 0.5

  11. Possible twins (If perfect twin)

  12. Hemihedral twinning • Intensities Iobs(h1) = (1–) I(h1) +  I(h2) Iobs(h2) =  I(h1) + (1–)I(h2) • Detwinning when   0.5 I(h1) = [ (1 –) Iobs(h1) – Iobs(h2)] / (1 – 2) I(h2) = [ – Iobs(h1) + (1 – ) Iobs(h2)] / (1 – 2)

  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 Perfectly twinned Untwinned Average over all resolution shells = 0.819

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

  16. Oxc Data Yeates & Fam ‘Merohedral Crystal Twinning Server’ http://www.doe-mbi.ucla.edu/Services/Twinning/

  17. Assigning space group

  18. = P3121 Crystallographic Self rotation analysis  = 180˚ Twinned data Data detwinned in P3121 NCS parallel to twinning axis

  19. Estimation of twin fraction The Yeates S(H) plot H = 0.5 –  H2 = (1 – 2)2 /3 H = [Iobs (h1) – Iobs (h2)] / [Iobs (h1) + Iobs (h2)] For acentric reflections S(H) = H / (1 – 2) Yeates, T.O. (1988). Acta Cryst. A44, 143-144.

  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. Perfect twin Untwinned Twinned data Detwinned  = 0.43 Detwinned  = 0.385 Local intensity statistics L = [Iobs(h1) – Iobs(h2)] / [Iobs(h1) + Iobs(h2)] where h1 and h2 are unrelated reflections For acentric data N(|L|) = |L| Padilla, J.E. & Yeates, T.O. (2003). Acta Cryst. D59, 1124-1130

  22. Estimated twin fraction

  23. Refinement • CNS- twin refinement protocol • Minimization • Bindividual • SHELXL- refinement of twin fraction Current values:  = 0.440 Rtwin= 14.9 Rfree= 16.1

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

  25. Density Thiamin diphosphate ADP Mg2+ 2FoFc- 1.5 FoFc- 4

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

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

  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

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