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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.
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