Data collection
References
Data were collected at 100 K using synchrotron radiation
and an ADSC Quantum 4 detector at beamline ID14-EH4
of the European Synchrotron Radiation Facility, Grenoble,
1 J. E. Baldwin and C. J. Schofield, in The biosynthesis of β-lactams,
ed. M. I. Page, Blackie, Glasgow, 1992, p. 1–78.
2 J. E. Baldwin, Spec. Publ. - R. Soc. Chem., 1985, 52, 62–85.
3 N. I. Burzlaff, P. J. Rutledge, I. J. Clifton, C. M. H. Hensgens,
M. Pickford, R. M. Adlington, P. L. Roach and J. E. Baldwin,
Nature, 1999, 401, 721–724.
4 J. E. Baldwin and M. Bradley, Chem. Rev., 1990, 90, 1079–1088.
5 J. E. Baldwin, R. M. Adlington, A. Basak, S. L. Flitsch,
A. K. Forrest and H.-H. Ting, J. Chem. Soc., Chem. Commun., 1986,
273–275.
6 J. E. Baldwin, R. M. Adlington, S. L. Flitsch, H.-H. Ting and
N. J. Turner, J. Chem. Soc., Chem. Commun., 1986, 1305–1308.
7 J. E. Baldwin, R. M. Adlington, L. G. King, M. F. Parisi,
W. J. Sobey, J. D. Sutherland and H.-H. Ting, J. Chem. Soc., Chem.
Commun., 1988, 1635–1637.
8 S. D. Abbott, 1991, D Phil Thesis, University of Oxford.
9 J. M. Ogle, I. J. Clifton, P. J. Rutledge, J. M. Elkins, N. I. Burzlaff,
R. M. Adlington, P. L. Roach and J. E. Baldwin, Chem. Biol., 2001,
8, 1231–1237.
10 P. J. Rutledge, N. I. Burzlaff, J. M. Elkins, M. Pickford, J. E. Baldwin
and P. L. Roach, Anal. Biochem., 2002, 308, 265–268.
11 P. L. Roach, I. J. Clifton, C. M. H. Hensgens, N. Shibata,
C. J. Schofield, J. Hajdu and J. E. Baldwin, Nature, 1997, 387, 827–
830.
12 P. L. Roach, I. J. Clifton, V. Fülöp, K. Harlos, G. J. Barton, J. Hajdu,
I. Andersson, C. J. Schofield and J. E. Baldwin, Nature, 1995, 375,
700–704.
13 D. H. Rich and J. P. Tam, J. Org. Chem., 1977, 42, 3815–3820.
14 S. Wolfe and M. G. Jokinen, Can. J. Chem, 1977, 57, 1388–1396.
15 J. E. Baldwin, S. R. Herchen, B. L. Johnson, M. Jung, J. J. Usher and
T. Wan, J. Chem. Soc., Perkin Trans. 1, 1981, 2253–2257.
16 J. C. Sheehan, J. Preston and P. A. Cruickshank, J. Am. Chem. Soc.,
1965, 87, 2492–2493.
France (IPNS:Fe():ACvG structure), or
a
300 mm
Mar Research Image Plate at Station 7.2 of the Synchrotron
Radiation Source, Daresbury, UK (IPNS:Fe():HMPen
structure).
Structure determination
Data was processed using MOSFLM19 and the CCP4 suite.20
Refinement was carried out using REFMAC5.21 The program
O22 was used for model building. Initial rigid body refinement
was followed by refinement of all atoms and individual atomic
temperature factors. Manual rebuilding of protein side-chains
was performed as necessary.
For the IPNS:Fe():ACvG structure, electron density
for the substrate ACvG was clearly visible throughout
refinement. Data from the oxygenated crystal gave a struc-
ture with active site electron density consistent with high
occupancy of a bicyclic penam molecule (HMPen), and
possible occupancy of the unreacted substrate ACvG at a low
level.
For the unexposed IPNS:Fe():ACvG structure, the initial
refinements of positional and individual isotropic temper-
ature factors were followed by refinement of individual
anisotropic temperature factors when the model was complete.
For the IPNS:Fe():HMPen structure, the final round of
refinement included refinement of TLS parameters.23 For
the HMPen molecule, coordinate restraints were generated
from selected analogous structures in the Cambridge Struc-
tural Database (CSD) of the Chemical Database Service
(CDS).24 Data collection and refinement statistics are given in
Table 1.
17 M. Bodanzky and A. Bodanzky, in The practice of peptide synthesis,
ed. K. Hafner, C. W. Rees, B. M. Trost, J. M. Lehn, R. v. R. Schleyer
and R. Zahruchik, Springer-Verlag, Berlin, 1984, p. 172.
18 P. L. Roach, I. J. Clifton, C. M. H. Hensgens, N. Shibata, A. J. Long,
R. W. Strange, S. S. Hasnain, C. J. Schofield, J. E. Baldwin and
J. Hajdu, Eur. J. Biochem., 1996, 242, 736–740.
19 A. G. W. Leslie, Acta Crystallogr., Sect D, 1999, 55, 1696–1702.
20 Collaborative Computational Project Number 4, Acta Crystallogr.,
Sect. D, 1994, 50, 760–763.
21 G. N. Murshudov, A. A. Vagin, A. Lebedev, K. S. Wilson and
E. J. Dodson, Acta Crystallogr., Sect. D, 1999, 55, 247–255.
22 T. A. Jones, J. Y. Zou, S. W. Cowan and M. Kjeldgaard,
Acta Crystallogr., Sect. A, 1991, 47, 110–119.
23 M. D. Winn, M. N. Isupov and G. N. Murshudov, Acta Crystallogr.,
Sect. D, 2001, 57, 122–133.
24 D. A. Fletcher, R. F. McMeeking and D. Parkin, J. Chem. Inf.
Comput. Sci., 1996, 36, 746–749.
Acknowledgements
We would like to thank the beamline scientists at the
SRS Daresbury and the ESRF Grenoble for their assis-
tance. This work was supported by the MRC, BBSRC and
EPSRC, UK. P.J.R. was supported by the Rhodes Trust, N.I.B.
by a German DAAD fellowship and P.L.R. by the Royal
Society.
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