Crystallographic findings on the internally uncoupled and near-rigor states of myosin: Further insights into the mechanics of the motor -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Himmel et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.202476799

Supporting Methods
Protein Preparation and Purification.
Subfragment 1 (S1) from scallop (Argopecten irradians) striated muscle myosin was prepared as described (1), and was complexed with either MgADP•BeF_x, MgAMPPNP (adenosine 5[prime]-[b,g-imido]triphosphate), MgATP-g -S (adenosine 5'-[g -thio]triphosphate), MgADP, or made nucleotide free. The S1-MgADP and S1-MgATP-g -S (3 mg/ml) each were rapidly cross-linked with para-phenyl dimaleimide (p-PDM) by treating the pH 7 buffered protein solution with 10-fold stoichiometry of 20 mM p-PDM dissolved in dimethylformamide for 2 min. at 20°C in the presence of 0.5 mM nucleotide. The reaction was stopped with 10 mM DTT. Intermolecularly cross-linked protein was removed by gel filtration. For nucleotide-free S1, the protein was dialyzed against 20 mM sodium cacodylate, pH 6.7/50 mM (NH₄)₂SO₄/2.0 mM MgCl₂/0.2 mM CaCl₂/0.1 mM EDTA/8% glycerol. The S1 complexes were precipitated with 55% ammonium sulfate and dialyzed against 20 mM MOPS, pH 7.0/80 mM NaCl/2 mM MgCl₂/0.2 mM CaCl₂/0.1 mM EDTA/3 mM NaN₃/0.5 mM DTT, 5 mg/ml leupeptin, and 10.5-50.0 mM nucleotide. Crystallization and Data Collection.
All the structures were obtained from intact, unmodified S1. In the case of the S1 complexes, 15 to 20 mg/ml protein were crystallized at 4°C in microseeded sitting drops that contained approximately equal volumes of protein and reservoir solution [67.5 mM Tris (pH 8.5-9.0), 6.2-6.6% wt/vol polyethylene glycol 20 K, and various concentrations of MgCl₂, SrCl₂, NaN₃, ethylene glycol, and nucleotide]. In the case of nucleotide-free S1, drops containing 35 mg/ml protein were combined with equal volumes of a precipitant solution containing 20 mM sodium cacodylate (pH 6.3), 50.0 mM (NH₄)₂SO₄, 2.0 mM MgCl₂, 8% glycerol, and 10% wt/vol polyethylene glycol 5 K monomethyl ether. This mixture was crystallized at 4.0°C in hanging drops over a reservoir solution in which the buffer, salt, and glycerol concentrations were double those of the precipitant solution. The crystals of the S1 complexes were triclinic, and the nucleotide-free crystals were monoclinic (Table 1). Data were collected at a temperature of 100 K at bnl and chess and were processed using hkl-denzo-scalepack (2) or scala (3) with the massage suite (N. Volkmann, unpublished material). Structure Determination and Refinement.
Phases were determined by molecular replacement and rigid body refinement with the program amore (4) using the scallop S1-MgADP structure (PDB ID code 1B7T) as an initial search model. Stepwise model building and torsional simulated annealing refinement were conducted using the o graphics package (5) and cns (6) with a bulk solvent correction (see Fig. 6 for a representative portion of the electron density map). Water molecules were added manually in the final stages of refinement and were only built in where they could be justified by hydrogen bonds and Fo-Fc electron density at or above the 3.0 s contour level. The Rfree values for all of the structures (calculated using a 5% data partition, Table 1) were within the average values for these resolutions (7). To compare the precision of the 2.3 Å S1-ADP•BeF_x structure with those of the lower resolution structures, we also calculated the coordinate error using the cross-validated Luzzati plot (7–9). Based on these calculations, the S1-ADP•BeFx structure had an average precision of 0.41 Å, similar to that of the S1-MgADP structure (1), while the other structures had a coordinate error of 0.5 Å to 0.7 Å. Some of the surface residues missing from the S1-MgADP structure (1) were built into the current structures, but there were still gaps in the heavy chain corresponding to disordered surface loops. In the nucleotide-free S1 structure, switch I was completely rebuilt based on a series of simulated annealing omit maps of portions of the loop, in which the Mg2+ ion and P loop residue T183 were also omitted.

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