Structure determination of Panulirus interruptus haemocyanin at 3.2 A resolution. Successful phase extension by sixfold density averaging.
The three-dimensional structure of Panulirus interruptus haemocyanin has been determined at 3.2 A resolution by X-ray diffraction techniques. Starting from a double isomorphous replacement map at 4 A resolution, the phases of the 32,709 reflections were first improved by six cycles of sixfold molecular averaging and solvent flattening. This also generated phase for 3078 reflections with no isomorphous replacement data. Next, phases for the reflections between 4.0 A and 3.2 A were obtained by a stepwise expansion procedure. In each expansion step 2000 to 3000 new reflections were added to the set of already phased reflections, followed by a few cycles of density averaging and solvent flattening at constant resolution. The eventual map at 3.2 A was calculated with 63,843 reflections with an average Sim weight of 0.75 and an overall R-factor of 23.5%. The polypeptide chain could be traced without any problems, while the dinuclear copper site, disulphide bridges and the first three moieties of the carbohydrate chain were clearly visible. Each subunit consists of three distinct domains. The first domain is mainly helical, containing one disulphide bridge and the carbohydrate chain. The second domain is also predominantly helical and contains the dinuclear copper site at its centre. The core of the third domain is an anti-parallel beta-barrel with the same topology as in the immunoglobulins and Cu,Zn-superoxide dismutase. This domain contains two disulphide bridges. Two long loops extend from the beta-barrel and have numerous interactions with the other two domains. The two copper ions are at approximately 3.7 +/- 0.25 A from each other and co-ordinated by six histidines, which are strictly conserved in all seven arthropodan haemocyanins with known amino acid sequences. No bridging protein ligand is present in the electron density distribution. Hydroxyl groups from tyrosines, which are invariant among arthropodan haemocyanins, are 17.4 A or more removed from the centre of the two copper ions. The closest Panulirus tyrosine hydroxyl is 10.6 A from the copper ions. So, it appears unlikely that tyrosine is involved in the co-ordination of the coppers either in the deoxy or in the oxy form of arthropodan haemocyanins.
W P
WP
Gaykema
A
A
Volbeda
W G
WG
Hol
D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D006433:Hemocyanins; D008121:Nephropidae; D010100:Oxygen; D011487:Protein Conformation; D014961:X-Ray Diffraction / Q000379:methods
D006433:Hemocyanins; D010100:Oxygen