Table 1.

Summary of selected crystallographic data

Crosslinked hemoglobin	α2β82CA82β	α2β82ND82β
Space group	P212121	P212121
Cell dimensions, Å	a = 86.8, b = 87.1, c = 97.6	a = 86.8, b = 87.1, c = 97.6
αβ dimers per ASU	2	2
Data collection
Resolution, Å	2.3	2.6
Number of observations/reflections	105,634/34,416	65,290/19,979
Rsym, %*	5.6	7.0
Refinement, Å	10.0–2.3	10.0–2.6
Completeness, %	99	78
R factor, %†	18.4	15.4
Number of atoms	4,576	4,578
Number of solvent molecules	143	51
Root-mean-squared deviations
Bond distances, Å	0.020	0.018
Bond angles, degrees	2.65	2.58

α₂β⁸²CA⁸²β and α₂β⁸²ND⁸²β were crosslinked in their deoxy forms by reacting human deoxyhemoglobin with the bis(methylphosphate) derivatives of either 4-carboxycinnamic acid (CA) or 2,6-napthalene dicarboxylic acid (ND), respectively (15, 16). α₂β⁸²CA⁸²β and α₂β⁸²ND⁸²β are crosslinked from the Nζ of β₁Lys-82 to the Nζ of β₂Lys-82. The negative charge of the methyl phosphate leaving groups serves to target the crosslinkers to the cationic 2,3-bis-phosphoglycerate binding pocket where the βLys-82 residues are located. Crystals of the carbonmonoxy form of α₂β⁸²CA⁸²β and α₂β⁸²ND⁸²β were grown at room temperature by the batch method of Perutz (17) from a solution of 2.4 M sodium-potassium phosphate, pH 6.7. These hemoglobins crystallize in space group P2₁2₁2₁, i.e., nonisomorphous with respect to unreacted carbonmonoxy hemoglobin. X-ray intensity data were collected at room temperature with an Area Detector Systems Corporation (ADSC) area detector using a Rigaku RU200-H rotating anode generator as the x-ray source (40 kV, 150 mA). These data were processed with the software provided by ADSC (19). The α₂β⁸²CA⁸²β hemoglobin was solved by molecular replacement using the merlot software package (18). The native carbonmonoxy αβ dimer (7), minus waters, was used as the starting model. Two large peaks in both the rotation and translation functions located the molecule and confirmed the presence of a tetramer in the asymmetric unit (ASU). Refinement with TNT (20) and difference Fourier syntheses located the cinnamyl crosslinker, which was fitted using frodo (21). Subsequent cycles of refinement to 2.3 Å followed until convergence. This structure, minus the crosslinker and waters, served as the starting model for the α₂β⁸²ND⁸²β structure. Crystals of α₂β⁸²ND⁸²β diffract to 2.3 Å resolution; however, they do not grow reproducibly, limiting our current data to 2.6 Å resolution. The napthalenyl crosslinker was located in a difference Fourier map, and the structure was refined to convergence. 

^*R_SYM = Σ |I_o − 〈I〉|/I_o, where I_o is the observed intensity, and 〈I〉 is the average intensity from multiple observations of symmetry-related reflections. 

^†R factor = Σ ∥F_obs| − |F_calc∥/Σ |F_obs|.