ACCURACY AND PRECISION IN PROTEIN CRYSTAL-STRUCTURE ANALYSIS - 2 INDEPENDENT REFINEMENTS OF THE STRUCTURE OF POPLAR PLASTOCYANIN AT 173-K

The structure of the copper protein plastocyanin from poplar leaves (P opulus nigra var. italica) at 173 K has been subjected to two independ ent refinements, using a single set of synchrotron X-ray data at 1.6 a ngstrom resolution. Energy-restrained refinement using the program ERE F resulted in lower root-mean-square deviations from ideal geometry (e .g. 0.011 angstrom for bond lengths) but a higher residual R (0.153) t han restrained least-squares refinement using the program PROLSQ (0.01 4 angstrom, 0.132). Electron-density difference maps in both refinemen ts provided evidence for disorder at some side chains and solvent atom s, and the PROLSQ refinement made allowance for this disorder. The num ber of solvent sites identified at the 4sigma(rho) level was 171 in th e EREF refinement and 189 in the PROLSQ refinement; 159 of the solvent sites are common to both refinements within 1 angstrom. The root-mean -square differences between the atomic positions produced by the two r efinements are 0.08 angstrom for C(alpha) atoms, 0.08 angstrom for bac kbone atoms and 0.12 angstrom for all non-H atoms (excluding six obvio us outliers) of the protein molecule. The two sets of Cu-ligand bond l engths differ by up to 0.07 angstrom, and the ligand-Cu-ligand angles by up to 7-degrees. At 173 K the volume of the unit cell is 4.2% small er than at 295 K. Greater order in the solvent region is indicated by the location of 79 more solvent sites, the identification of extensive networks of hydrogen-bonded rings of solvent molecules, and a general decrease in the thermal parameters. Within the unit cell, the protein molecules are significantly translated and rotated from their positio ns at ambient temperature. An important structural change at low tempe rature is a 180-degrees flip of the peptide group at Ser48-Gly49. Near ly all other significant differences between the structures of the pro tein at 173 and 295 K occur at exposed side chains. If the backbone at oms in the 173 and 295 K structures are superposed, excluding atoms in volved in the peptide flip, the root-mean-square difference between th e positions of 393 atoms is 0.25 angstrom. Two internal water molecule s, not included in previous descriptions of poplar plastocyanin, have been located. The plastocyanin Cu-site geometry at 173 K is not signif icantly different from that at 295 K. If plastocyanin undergoes a chan ge in Cu-site geometry at low temperature, as has been suggested on th e basis of resonance Raman spectroscopic evidence, then the change is not detected within the limits of precision of the present results.