Structure
Structural Analysis of SpuA
EOM (Bernado et al., 2007) was applied to the SAXS data as follows.
The polypeptide region (ꢀaa 435–438) between the linker domain and the
N-domain of the catalytic module was treated as a ‘‘hinge,’’ and a random
pool of 10,000 conformers was generated around the flexible hinge using
RANCH from which the genetic algorithm GAJOE selected the ensemble fitting
the experimental data. EOM modeling of native SpuA was done using data
collected on the 2.95 mg/ml concentration of SpuA. Both data sets were
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analyzed over a Q range of ꢀ0.06–0.5 AÀ1. The number of ensembles to
Brunger, A.T. (1992). Free R value: a novel statistical quantity for assessing the
˚
accuracy of crystal structures. Nature 355, 472–475.
compare to the experimental data was set at 50, and the number of genera-
tions the genetic algorithm used to optimize the ensemble was 1000, with
20 crossings per generation and 50 repetitions. c values were determined
for the ensembles during EOM modeling and separately for the individual
ensemble models using the program CRYSOL (Svergun et al., 1995).
Davies, G.J., Wilson, K.S., and Henrissat, B. (1997). Nomenclature for sugar-
binding subsites in glycosyl hydrolases. Biochem. J. 321, 557–559.
Eckhart, L.J. (1982). Kinetic measurement of alpha-amylase by a P-nitrophenyl
maltopentaoside method using the American Monitor Kda. Clin. Chem. 28,
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ACCESSION NUMBERS
Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular
graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132.
Coordinates and structure factors have been deposited with the PDB codes of
2ya1 for SpuA with maltotetraose, 2ya0 for SpuADCBM, and 2ya2 for
SpuADCBM in complex with G1M.
Ficko-Blean, E., Gregg, K.J., Adams, J.J., Hehemann, J.H., Czjzek, M., Smith,
S.P., and Boraston, A.B. (2009). Portrait of an enzyme, a complete structural
analysis of
a multimodular b-N-acetylglucosaminidase from Clostridium
perfringens. J. Biol. Chem. 284, 9876–9884.
SUPPLEMENTAL INFORMATION
Fischer, H., Neto, M.D., Napolitano, H.B., Polikarpov, I., and Craievich, A.F.
(2010). Determination of the molecular weight of proteins in solution from
Supplemental Information includes Supplemental Experimental Procedures
a
single small-angle X-ray scattering measurement on
a relative scale.
J. Appl. Crystallogr. 43, 101–109.
Franke, D., and Svergun, D.I. (2009). DAMMIF, a program for rapid ab-initio
shape determination in small-angle scattering. J. Appl. Crystallogr. 42,
342–346.
ACKNOWLEDGMENTS
We acknowledge Alexey Kikhney and all other staff members for help on the
SAXS beamline X33 at the DORIS-III storage ring at DESY (Deutsches Elektro-
nen-Synchrotron). This work was supported by grants from the British
Columbia Lung Association (to A.B.B.), the Canada Institutes of Health
Research (to A.B.B. and D.J.V.), the Natural Sciences and Engineering
Research Council of Canada (NSERC; to R.D.B.), and the National Health
and Medical Research Council of Australia (NHMRC; to J.C.P.). A.LvB. and
M.A.H. are supported by doctoral fellowships from NSERC and the Michael
Smith Foundation for Health Research (MSFHR). B.P. is supported by
a MSFHR Post-Doctoral Fellowship. J.-H.H. was supported by a Marie-Curie
doctoral Fellowship. J.C.P. is a NHMRC Australia Fellow. D.J.V. is a Canada
Research Chair in Chemical Glycobiology and a MSFHR Career Scholar.
A.B.B. is a Canada Research Chair in Molecular Interactions and a MSFHR
Career Scholar.
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mimicry of the transition state. Org. Biomol. Chem. 8, 305–320.
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(2009). Group B streptococcus pullulanase crystal structures in the context of
a novel strategy for vaccine development. J. Bacteriol. 191, 3544–3552.
Hammel, M., Fierobe, H.P., Czjzek, M., Finet, S., and Receveur-Brechot, V.
(2004). Structural insights into the mechanism of formation of cellulosomes
probed by small angle X-ray scattering. J. Biol. Chem. 279, 55985–55994.
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ence and invasion of epithelial cells. Infect. Immun. 73, 4653–4667.
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Streptococcus pneumoniae virulence factors. Mol. Microbiol. 45, 1389–1406.
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Received: January 5, 2011
Revised: February 22, 2011
Accepted: March 3, 2011
Published: May 10, 2011
Hyams, C., Camberlein, E., Cohen, J.M., Bax, K., and Brown, J.S. (2010). The
Streptococcus pneumoniae capsule inhibits complement activity and neutro-
phil phagocytosis by multiple mechanisms. Infect. Immun. 78, 704–715.
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650 Structure 19, 640–651, May 11, 2011 ª2011 Elsevier Ltd All rights reserved