During effor
ts
to crys
tallize
the enzyme 2,4-dihydroxyace
to
phenone dioxygenase (DAD) from
Alcaligenes s
p. 4HAP, a small number of s
trongly diffrac
ting
pro
tein crys
tals were ob
tained af
ter
two years of crys
tal grow
th in one condi
tion. The crys
tals diffrac
ted synchro
tron radia
tion
to almos
t 1.0 Å resolu
tion and were, un
til recen
tly, assumed
to be formed by
the DAD
pro
tein. However, when ano
ther crys
tal form of
this enzyme was even
tually solved a
t lower resolu
tion, molecular re
placemen
t using
this new s
truc
ture as
the search model did no
t give a convincing solu
tion wi
th
the original a
tomic resolu
tion da
ta se
t. Hence, i
t was considered
tha
t these crys
tals migh
t have arisen from a
pro
tein im
puri
ty, al
though molecular re
placemen
t using
the s
truc
tures of common crys
talliza
tion con
taminan
ts as search models again failed. A scri
pt to
perform molecular re
placemen
t using
MOLREP in which
the firs
t chain of every s
truc
ture in
the PDB was used as a search model was run on a mul
ti-core clus
ter. This iden
tified a number of
prokaryo
tic
phos
pha
te-binding
pro
teins as scoring highly in
the
MOLREP peak lis
ts. Calcula
tion of an elec
tron-densi
ty ma
p a
t 1.1 Å resolu
tion based on
the solu
tion ob
tained wi
th PDB en
try
ttp://scripts.iucr.org/cgi-bin/explore.cgi?pdbid=2q9t" rel="references:http://scripts.iucr.org/cgi-bin/explore.cgi?pdbid=2q9t">2q9t allowed mos
t of
the amino acids
to be iden
tified visually and buil
t in
to
the model. A
BLAST search
then indica
ted
tha
t the molecule was mos
t probably a
phos
pha
te-binding
pro
tein from
Stenotrophomonas maltophilia (UniPro
t ID B4SL31; gene ID Smal_2208), and fi
tting of
the corres
ponding sequence
to
the a
tomic resolu
tion ma
p fully corrobora
ted
this. Pro
teins in
this family have been linked
to
the virulence of an
tibio
tic-resis
tan
t s
trains of
pa
thogenic bac
teria and wi
th biofilm forma
tion. The s
truc
ture of
the
S. maltophilia pro
tein has been refined
to an
R fac
tor of 10.15% and an
Rfree of 12.46% a
t 1.1 Å resolu
tion. The molecule ado
pts
the
ty
pe II
peri
plasmic binding
pro
tein (PBP) fold wi
th a number of ex
tensively elabora
ted loo
p regions. A fully dehydra
ted
phos
pha
te anion is bound
tigh
tly be
tween
the
two domains of
the
pro
tein and in
terac
ts wi
th conserved residues and a number of helix di
poles.