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   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).
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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
   secondary structures listed in the text are composed of the following
   residues: β15, 187–189; β18, 200–204; β25, 240–245; β26, 258–260; β28,
   267–270; β32, 302–304; β35, 378–381; β36, 383–386; β39, 434–436. B,
   ribbon representation of the structure. The β-helix core, domain A, and
   domain B are shown, respectively, in blue, gold, and red. The small T1
   extension, containing an antiparallel sheet (β16-β17) and an α-helix
   (α2), is shown ingreen. The red, yellow, andgreen spheres represent
   sodium, calcium, and chloride ions, respectively. Figs. [89]3 and [90]4
   were prepared using Molscript ([91]50).

   The most striking difference between ι-carrageenase (Fig. [92]3) and
   the 11 other β-helix proteins of known structure is the presence, in
   the C-terminal region, of two large additional domains (both 67
   residues long). Domain A (residues 307–373) replaces the T1 turn
   between strands β32 and β35 (see legend of Fig. [93]3 A for
   definition). Half of this domain (residues 314–333 and 341–350) could
   not be built as no clear electron density was observed. The visible
   part of domain A features a sheet of two short antiparallel β-strands,
   β33 and β34, edged by one α-helix (residues 358–367) and has an average
   B value (37.8 Å^2) twice that of the β-helix core (17.8 Å^2). At the
   border of the visible part of domain A is a hydrophobic surface,
   suggesting that the nonvisible residues complete a globular-shaped
   domain. This domain is weakly bound to the β-helix by only four
   hydrogen bonds (Glu^310 O-Lys^252 NZ, Asp^358OD1-Ser^442 OG, Asp^358
   OD2-Lys^443 N, and Asp^362 OD1-Tyr^444 OH). Moreover, the visible part
   of domain A makes no contact with neighboring molecules and is located
   in a large solvent channel in the crystal. Domain B consists of
   residues 387–430, located on a T3 turn that connect strands β36 and
   β39, and the C-terminal extension (residues 469–491). Mainly composed
   of long loops, it also features an antiparallel β-sheet (β37-β38), a
   3[10] helix (residues 395–402), and a one-turn α-helix at the C
   terminus. This domain, also globular in shape, is folded around an
   independent hydrophobic core. In contrast to domain A, this bulky
   structure is characterized by many side chain-side chain and side
   chain-main chain hydrogen bonds, both within the domain and with the
   β-helix. These hydrogen bonds have a clear stabilizing effect on domain
   B since its average B value (22.0 Å^2) is close to that of the β-helix
   core (17.8 Ã…^2).

   Each strand of PB2a is connected to PB2b by an asparagine residue in a
   left-handed α-helix conformation. Residues in the α[L]conformation are
   also seen in the T2 turns in the structure. The presence of these
   residues results in sharp bends of about 100° in the polypeptide chain
   without disrupting the hydrogen bond pattern of the parallel β-strands.
   PB1 also presents a striking repetition of a structural irregularity:
   in each strand, one residue is in a right-handed helix conformation,
   thus creating an alignment of nine β-bulges in the groove. With the
   exceptions of Ser^186 and the Lys^464-Thr^465 β-bulge in the C
   terminus, which are accessible to the solvent, all side chains in these
   β-bulges are aliphatic amino acid residues and point toward the
   hydrophobic core of the β-helix.

   The turns and loops between the β-strands are of different sizes and
   have different conformations. Whereas almost all of the T2 turns
   consist of a single residue in the α[L] conformation, the T1 and T3
   turns are longer and more irregular, their sizes ranging from 1 to 12
   and from 2 to 8 residues, respectively. At the N-terminal edge of the
   β-helix, the T3 loops form a bulky protrusion above PB1. On the
   opposite side of this β-sheet, the T1 turn between strands β15 and β18
   folds into a β-sheet of two antiparallel strands (β16 and β17), and the
   T1 turn between β25 and β26 folds into a short α-helix. Strands β16 and
   β17 and this short α-helix form a domain that is stabilized by a
   hydrophobic core (Val^191, Leu^198, Leu^250, Met^251, and Tyr^254) and
   hydrogen bonds between Glu^193OE2-Tyr^254 OH and Leu^198 O-Gln^256OE1.
   At the surface, two hydrophilic networks (Arg^136-Asp^166-Arg^194 and
   Arg^202-Trp^200-Asp^227-Arg^260-Gly^257-Gly^258) firmly bind this
   domain to the β-helix core; these amino acids are also present in Z.
   galactanovorans ι-carrageenase. On each edge of β-sheet PB1, the
   N-terminal T1 and T3 extensions and the protruding domains A and B in
   the C-terminal region create a long deep cleft. This large channel,
   about 50 Ã… long and 10 Ã… wide, is probably the cleft that binds
   ι-carrageenan chains.

   A. fortis ι-carrageenase contains four disulfide bridges. The first,
   Cys^269-Cys^298, is located within the β-helix core, connecting strand