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 §3§ Methods §3§

 §4§ Protein purification §4§

   AChBP was cloned into expression vector pPIC9 (without residue Leu 1)
   and overexpressed in yeast, Pichia pastoris GS115, according to the
   Invitrogen manual. After 4 days of induction the medium was collected,
   concentrated and dialysed against standard buffer (20 mM Tris–HCl
   buffer (pH 8.0), 150 mM NaCl and 0.02% (w/v) NaN[3]). The protein was
   purified by anion exchange (Poros50 HQ, MonoQ), and gel filtration
   (Superdex 200). It was dialysed against 50 mM HEPES buffer (pH 7.0)
   with 0.02% NaN[3] and concentrated to approx 20 mg ml^-1. N-terminal
   sequencing revealed that part of the pPIC9-encoded signal sequence is
   retained, before residue 2 (sequence EAEAYVEF). The experimental
   relative molecular mass was 26,544 (MALDI), approx 2K more than the
   calculated mass based on the sequence (24,649), owing to glycosylation
   at position Asn 66, as confirmed by deglycosylation experiments (data
   not shown).

 §4§ Crystallization §4§

   We grew the crystals at room temperature using the hanging-drop vapour
   diffusion technique. All drops contained 2  micro l of protein
   (10 mg ml^-1) and 2  micro l of reservoir solution (9–11% (w/v) PEG
   4000, 100 mM HEPES (pH 7.0), 50–200 mM CaCl[2] and 0.02% NaN[3]).
   Depending on the batch of protein and the CaCl[2] concentration, we
   obtained three crystal forms. Orthorhombic and monoclinic crystals
   appeared under high CaCl[2] concentration and were frequently twinned.
   The orthorhombic crystals (P2[1]2[1]2[1]) have the following cell
   constants: a = 120.6 Ã…, b = 137.0 Ã…, c = 161.5 Ã…, with two pentamer
   molecules per asymmetric unit (asu). The monoclinic crystals (P2[1])
   are very similar in morphology to the orthorhombic ones, but gave lower
   resolution data ( approx 3.3 Ã…), with the following cell constants: a =
   121.1 Å, b = 162.1 Å, c = 139.4 Å, beta = 90.13°, and four pentamers
   per asu. The tetragonal crystal form (P4[2]2[1]2) was obtained from a
   solution containing 11.5 (w/v) PEG 4000, 100 mM HEPES (pH 7.0), 150 mM
   CaCl[2] and 0.02% (w/v) NaN[3]. They have the following cell constants:
   a = b = 141.66 Ã…, c = 120.83 Ã… and one pentamer per asu. For MAD
   experiments, orthorhombic and monoclinic crystals were soaked in mother
   liquor solution containing 5 mM and 10 mM trimethyl-lead acetate (MePb)
   respectively for 5 days. Before flash-cooling, all crystals were
   gradually equilibrated against mother liquor with 30% glycerol.

   In all three space groups AChBP forms a decamer structure with 52
   symmetry, where 2 pentamers have contact with each other through a
   calcium-binding site, at the 'top' of the alpha 1 helix. This binding
   site (Asp 2 and Asp 5 from two protomers) is not conserved in the

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   removed by DEAE anion exchange chromatography and gel filtration on a
   Sephacryl S100 HR column, and the Fab was purified further by ion
   exchange chromatography on a mono Q FPLC column by a NaCl gradient in
   20 mM ethanolamine buffer at pH 9.3.

   Crystals were grown at 4¡C by using the hanging-drop procedure in wells
   containing 1 ml of 16% (vol/vol) polyethylene glycol 4000, 3% (vol/vol)
   dioxan, 20% (vol/vol) glycerol, 0.2 M ammonium sulfate, 5 mM strontium
   chloride, and 20 mM sodium acetate (final pH 5). Drops consisting of a
   2-μl aliquot of a protein solution with hapten (0.25 mM hapten and 11.6
   mg of Fab per ml in 0.15 M NaCl/0.05% NaN[3]) were mixed with 2 μl of
   the well solution. Despite the simultaneous growth of thin needles and
   polyhedral-shaped crystals, this procedure yielded, in some drops,
   monocrystals of dimensions up to 0.7 × 0.45 × 0.35 mm^3.

 ¤3¤ X-Ray Data Collection and Structure Determination. ¤3¤

   Diffraction data were recorded by using one crystal kept at 4¡C on the
   W32 station of the Laboratoire pour l'Utilisation du Rayonnement
   ElectromagnŽtique (Orsay, France) synchrotron with a MAR Image Plate
   system. Data were processed with denzo and scalepack ([55]16), and