Catrine Berthold, PhD student
Supervisor Prof. Ylva Lindqvist
Molecular Structural Biology
Medical Biochemistry and Biophysics
Karolinska Institutet
Structure of
Oxalyl-CoA decarboxylase
solved from
a hemihedrally twinned crystal
Oxalate
O
O
O
O
Characteristics:
• Highly oxidized
• Strong chelator of
cations
Toxic to most
life forms
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
Oxalobacter formigenes
• Depends on oxalate as unique energy
and carbon source
• Colonizes the gastrointestinal tract of
most vertebrates
• Symbiotic relationship with mammals
Ricagno, S. et al. (2003)
EMBO J. 22, 3210-3219.
Oxalate2-
Formate1O
H
O
O
O
H
Formyl-CoA
O
1
SC o A
O
O
Formyl-CoA transferase
O
Oxalyl-CoA decarboxylase
Oxc
S Co A
O
CO
2
2
Frc
O
Oxalyl-CoA
Oxc crystal form 1
Space group:
Resolution:
P42212
4.1 Å
Molecular Replacement:
Program: MolRep
Template: (23% ident.) Modified
Acetohydroxyacid synthase
CC:
0.528
Incomplete model
Oxc crystal form 2
Space group:
P3121
Resolution:
1.73 Å
Twin fraction:
~ 0.44
Data collection statistics
High resolution pass
Beamline
ID23-1 ESRF
Wavelength (Å)
Resolution (Å)
Low resolution pass
0.9757
29.24-1.73 (1.82-1.73)
62.02-2.32 (2.45-2.32)
Space group
P3121
a, b (Å)
c (Å)
124.3
151.7
No. observations
1004713 (145215)
272295 (10045)
No. unique
140751 (20434)
41398 (1360)
Rsym (%)
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 (Å2)
21.1
39.4
What is twinning?
• Non-merohedral (epitaxial)
• Merohedral
- Hemihedral - two domains
Dauter, Z. (2003) Acta Cryst. D59, 2004-2016.
Merohedral twinning
Single untwinned crystal with two domains
= twin fraction
Hemihedrally twinned crystal
Perfect  = 0.5, Partial  < 0.5
Possible twins
(If perfect twin)
True point group Twin operation
hkl related to
Apparent
point group
3
2 along a,b
h, -h-k, -l
321
2 along a*,b*
h+k, -k , -l
312
2 along c
-h, -k , l
6
4
2 along a,b,a*,b*
h, -k , -l
422
6
2 along a,b,a*,b*
h, -h-k, -l
622
321
2 along c,a*,b*
-h, -k , l
622
312
2 along c,a,b
-h, -k , l
622
23
4 along a,b,c
k, -h , l
432
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)
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
Wilson Ratio
Perfectly twinned
Untwinned
Average over all resolution shells = 0.819
Space group ambiguity
Apparent symmetry:
Possible true
point symmetries:
6
622
2 along c, a*, b*
321
312
3
Oxc Data
P622
P6
P321
P312
Rmerge
0.079
0.077
0.060
0.078
Nmol/asym unit
1
2
2
2
Twin fraction (α)
0.00
0.48
0.44
0.48
Yeates & Fam ‘Merohedral Crystal Twinning Server’
http://www.doe-mbi.ucla.edu/Services/Twinning/
Assigning space group
MR statistics:
P62
P64
P3121
P3221
P3112
P3212
Rfactor
55.4
55.3
59.7
61.0
50.8
50.7
Corr. coef.
20.2
19.9
23.9
18.2
21.0
20.4
10 cycles of Rigid body refinement in CNS
Rtwin
44.6
44.2
43.5
44.5
44.6
44.1
Rfree
43.4
44.5
41.9
42.6
46.6
47.0
Self rotation analysis
 = 180˚
Twinned data
= P3121 Crystallographic
Data detwinned in P3121
NCS parallel to twinning axis
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.
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
Local intensity statistics
Twinned data
Detwinned  = 0.43
Detwinned  = 0.385
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
Estimated twin fraction
Britton plot
Yeates statistics
H
H2
Local intensity statistics

0.43
0.43
0.43
0.42
0.39
Refinement
• CNS- twin refinement protocol
– Minimization
– Bindividual
• SHELXL- refinement of twin fraction
Current values:
 = 0.440
Rtwin= 14.9
Rfree= 16.1
Rtwin = | [ F2calc(h1) + F2calc(h2)]1/2 - Fobs(h1) | /  | Fobs(h1) |
Structure- Tetramer
Oxalyl-CoA decarboxylase (Oxc)
Density
Thiamin
diphosphate
ADP
2FoFc- 1.5
FoFc- 4
Mg2+
NCS promotes twinning
90˚
6.5Å shift
Twin domain 1
Twin domain 2
Twin domains
Twin axis- 180˚ along c
Twin domain 1
Twin domain 2
Monomer B in blue
Acknowledgements
Supervisors:
Prof. Ylva Lindqvist & Prof. Gunter Schneider
Collaborators:
Prof. Nigel Richards
Patricia Moussatche
(Unv. of Florida)
Special thank to all my coworkers in the Molecular
Structural Biology group at
the Karolinska Institute