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   the native data set to 1.6 Ã… with the maximum likelihood target
   function. The program was set up to automatically compute a
   cross-validated Ï‚[a] estimate and the weighting scheme between the
   x-ray refinement target and the geometric energy function. Corrections
   for a flat bulk solvent and for anisotropy in the data were also
   applied. The Ï‚[a] weighted maps obtained from the subsequent refinement
   models were used for further model building. The first group of water
   molecules was added when peaks in the 2F [o] −F [c] density were >2 ς
   and had a stereochemistry compatible with at least one hydrogen bond
   with a protein atom or another water molecule. In the final stages, the
   Ï‚ cut-off was reduced to 1.0 Ï‚, and water molecules with a B factor >60
   Ã…^2 were removed from the model. The final model refined at 1.6 Ã… has
   an R[work] of 20.7% and an R[free] of 22.3% ([66]25) and consists of
   6,840 protein atoms, 1,110 water molecules, six glycerol molecules, and
   six calcium, six sodium, and two chloride ions. All these ions have an
   occupancy of 1, and their B factors refine to a value close to
   neighboring protein atom B factors. The stereochemistry of the final
   structure was evaluated using the PROCHECK program ([67]26).

 §5§ Calcium-dependent Activity Test and Proteolytic Digestion §5§

   ι-Carrageenase activity was assayed as follows. Three enzyme solutions
   were prepared by diluting a stock solution of the purified enzyme (6
   mg/ml) in 100 mm Tris-HCl, pH 7.2, 200 mm NaCl and adding either 5 mm
   EGTA or 5 mm CaCl[2]. Aliquots (100 μl) were incubated for 15 min at
   40 °C with 2 ml of substrate solution consisting of 0.125% (w/v)
   ι-carrageenan, 50 mm Tris-HCl, pH 7.2, 100 mm NaCl with or without 5 mm
   CaCl[2], and the reaction mixture (200 μl) was assayed for reducing
   sugars ([68]27) using boiled enzyme blanks. One unit of enzyme activity
   is defined as the amount of enzyme that produces an increase of 0.1 A
   [237 nm]/min in the reducing sugar assay.

   Purified bovine pancreas trypsin (T1426, 10,000N-benzoyl-l-arginine
   ethyl ester units/mg of protein) was purchased from Sigma. Limited
   proteolysis of ι-carrageenase (6 mg/ml in 100 mm Tris-HCl, pH 7.2, 200
   mm NaCl) was performed using trypsin/ι-carrageenase ratios of 1:100 and
   1:20 (w/w) in the presence or absence of 5 mm EGTA or 5 mm CaCl[2]. The
   samples were incubated for 1 h at 20 °C, and then the reaction was
   stopped by adding SDS loading buffer and boiling the samples for 5 min
   at 100 °C. The samples were then loaded onto a 15% SDS-polyacrylamide
   gel for electrophoresis, and the gels were stained with Coomassie Blue.
   [69]Previous Section[70]Next Section

 §2§ RESULTS §2§

 §5§ The Overall Structure of A. fortis ι-Carrageenase §5§

   The three-dimensional crystal structure of ι-carrageenase lacking the
   signal peptide (residues 1–27) was determined at 2.3 Å resolution by
   the multiple anomalous diffraction method using a crystal of a
   Se-Met-substituted form of the enzyme ([71]28). After phase extension
   with a native data set at higher resolution, a high quality electron
   density map was obtained (Fig. [72]2) allowing building and refinement
   of the model at 1.6 Ã… resolution. The crystallographic statistics are
   shown in Table [73]I. The asymmetric unit contains two mature
   ι-carrageenase molecules, each containing amino acids 28–491. Residues
   314–334 and 341–350 for molecule A and residues 313–334 and 341–351 for
   molecule B are not visible in the 2F [o] − F [c] electron density map
   and are presumed to exist in disordered or highly flexible
   conformations. Superposition of molecules A and B reveals that the Cα
   atoms overlay with a root mean square deviation of 0.21 Ã….
   Approximately 10 residues in each molecule presented clear alternate
   conformations. The need to refine the occupancy for terminal atoms of
   several residues, such as aspartate, glutamate, or methionine, suggests
   that a fraction of the protein population in the crystal has been
   subjected to radiation damage ([74]29).
   [75]Figure 2
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   Figure 2

   Solvent-flattened multiple anomalous diffraction electron density map
   at 1.6 Ã… resolution. Map contoured at 2.0 Ï‚ of the N-terminal
   calcium-binding hairpin loop. Calcium ion and water molecules are
   indicated as yellow andred spheres, respectively. The oxygen, nitrogen,
   and carbon atoms in the protein are shown in red, blue, andyellow,
   respectively. This figure was created using O ([79]23).
   View this table:
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   Table I

   Data reduction, phasing, and refinement statistics

   The core of ι-carrageenase is folded into a right-handed parallel
   β-helix of 10 complete turns (Fig. [82]3). This fold was first
   encountered in pectate lyase C from Erwinia chrysanthemi ([83]30). The
   lyase structure consists of three parallel β-sheets, PB1, PB2, and PB3.
   PB2 and PB3 form planar surfaces almost perpendicular to each other,
   while PB1 is in the form of a groove. In the lyase structure, the turns
   or loops (depending on the number of amino acids inserted between
   consecutive β-helical strands) are referred to as T1 (PB1-PB2), T2
   (PB2-PB3), and T3 (PB3-PB1). In β-helix proteins, the assignment of
   secondary structure elements is based on the DSSP algorithm ([84]31)
   with the additional criterion that any residues with (Φ, ψ) angles in
   the left-handed α-helix region are not included in the β-strand. Based
   on these rules, PB2 can be divided into two parts, and the
   ι-carrageenase β-helix consists of four parallel β-sheets, PB1, PB2a,
   PB2b, and PB3, composed respectively of 10, 5, 11, and 10 β-strands.
   These strands are relatively short with an average number of 4.0, 2.4,
   4.1, and 4.0 residues, respectively. Interestingly, like almost all
   β-helix proteins, ι-carrageenase contains in its N-terminal region an
   amphipathic α-helix (residues 66–77) that shields the hydrophobic core
   of the β-helix from the solvent.
   [85]Figure 3
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   Figure 3

   Folding of A. fortisι-carrageenase. A, stereo view of the Cα trace of
   the protein. The N terminus, C terminus, and every 20th residue are
   labeled, while every 10th residue is marked with ablack dot. The