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We have applied the method of simulated annealing to the refinement of the 3 A
resolution crystal structure of the influenza virus hemagglutinin glycoprotein,
using the program X-PLOR. Two different methods were introduced into X-PLOR to
treat the non-crystallographic symmetry present in this and in other crystal
structures. In the first, only the unique protomer atoms are refined; by
application of the non-crystallographic symmetry operators to the protomer
atoms, the X-ray structure factor derivatives are effectively averaged, and a
non-bonded energy term models the interactions of the protomer with its
neighbors in the oligomer without explicit refinement of the other protomers in
the crystallographic asymmetric unit. In the second method, the entire
asymmetric unit is refined, but an effective energy term is added to the
empirical energy that restrains symmetry-related atomic positions to their
average values after least-squares superposition. Several other modifications
and additions were made to previously published X-PLOR protocols, including
weighting of the X-ray terms, maintenance of the temperature of the molecular
dynamics simulation, treatment of charged groups, changes in the values of
certain empirical energy parameters, and the use of N-linked carbohydrate
empirical energy parameters. The hemagglutinin refinement proceeded in several
stages. An initial round of simulated annealing of the monomer was followed by
rigid-body refinement of the 3-fold non-crystallographic symmetry axis position
and a second round of monomer refinement. A third round was performed on the
trimer using non-crystallographic symmetry restraints in all regions except
those in lattice contacts showing obvious derivations from 3-fold symmetry. The
refinement was completed with several rounds of conventional positional and
isotropic temperature factor refinement needed to correct bad model geometry
introduced by high-temperature molecular dynamics in regions of weak electron
density. This structure was then used as the basis for refinement of three
crystallographically isomorphous hemagglutinin structures, including complexes
with the influenza virus receptor, sialic acid. Model geometry comparable to
well-refined high-resolution structures was obtained with relatively little
manual intervention, demonstrating the ability of simulated annealing refinement
to produce highly idealized structures at moderate resolution.
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