  Rigidity analysis using the "pebble game" has been applied to protein crystal
structures to obtain information on protein folding, assembly and t he
structure-function relationship. However, previous work using this technique
has not made clear how the set of hydrogen-bond constraints included in the
rigidity analysis should be chosen, nor how sensitive the results of rigidity
analysis are to small structural variations. We present a comparative study in
which "pebble game" rigidity analysis is applied to multiple protein crystal
structures, for each of six differen t protein families. We find that the
mainchain rigidity of a protein structure at a given hydrogen-bond energy
cutoff is quite sensitive to small structural variations, and conclude that the
hydrogen bond constraints in rigidity analysis should be chosen so as to form
and test specific hypotheses about the rigidity o f a particular protein. Our
comparative approach highlights two different characteristic patterns ("sudden"
or "gradual") for protein rigidity loss as constraints are re moved, in line
with recent results on the rigidity transitions of glassy networks.
