BACKGROUND
pfam is a comprehensive collection of protein domains and families, with a range of well-established information including genome annotation.
each family in pfam is represented by two multiple sequence alignments and two profile-hidden markov models   <cit> .
pfam has two large series of functionally uncharacterized families, known as domains of unknown function  and uncharacterized protein families .
dufs are families that have been created by pfam whereas upfs are those created by swiss-prot and added to pfam  <cit> .
the protein structure initiative- <dig> has undertaken the task of structurally characterizing all pfam families that have no structural representation.
the pfam protein family duf <dig>  consists of  <dig> proteins of unknown function,  <dig> from bacteria and  <dig> from archaebacteria  <cit> .
herein, we report the first crystal structures of duf <dig> family members, including q9rxe <dig> from deinococcus radiodurans and q9l0t <dig> streptomyces coelicolor and show that they are remarkably similar to pfam family duf <dig>
RESULTS
the structure of 10093b was determined to  <dig> Å resolution using selenium-sad .
the final refined model of 10093b contains  <dig> protomers,  <dig> sulfate ions, and  <dig> water molecules.
the final refined model of 10093f  contains  <dig> protomers and  <dig> water molecules.
despite low sequence identity , the polypeptide chain structures are very similar .
the two structures superimpose well with a rmsd of  <dig> Å for  <dig> α-carbon atomic pairs, excluding a loop region between val <dig> to ser <dig> in 10093b that does not occur in 10093f .
this region is absent in all other sequences shown in figure  <dig>  except in thermus thermophilus .
values for the highest resolution shell are given within parentheses.
1rmerge = Σ|ii-⟨i⟩|/Σ|ii| where ii is the intensity of the ith measurement, and ⟨i⟩ is the mean intensity for that reflection.
2phasing power and 3fom  as defined in sharp.
4rfactor = Σ||fobs|-|fcalc||/Σ|fobs| where |fcalc| and |fobs| are the calculated and observed structure factor amplitudes, respectively.
notwithstanding the similarity of the two polypeptide chain folds, mr attempts with various 10093b-derived search models were not successful.
molecular replacement may have failed because of low sequence identity and/or due to the presence of multiple protomers within the asymmetric unit.
biologically active units
10093b
eight monomers a, b, c, d, e, f, g, and h found in the asymmetric unit form four tightly packed dimers .
each dimer pair superimposes very well on the remaining three with rmsds =  <dig> Å– <dig> Å for all α-carbon atomic pairs.
a dimer interface analysis performed using pdbsum  <cit>  demonstrated that each pair buries ~ <dig> Å <dig> of solvent accessible surface area , a value higher than would be found typically in interacting surfaces for a protein of this size  <cit> .
at least  <dig> residues from each half of the dimer participate in intermolecular interactions.
there are  <dig> direct hydrogen bonded interactions between the protein atoms across the dimer interface.
such a tight dimer interface suggests that the 10093b dimer is functional, which is supported by the results of analytical gel filtration .
10093f
four monomers a, b, c, and d found in the crystallographic asymmetric unit occur as two dimers , albeit with an intermolecular packing arrangement that differs from that seen for 10093b.
each observed 10093f dimer pair buries ~ <dig> Å <dig> of solvent accessible surface area, which is not thought to be significant for a protein of this size.
moreover, the gel-filtration analysis  revealed that 10093f is a monomer in solution.
in the 10093b dimer, glu <dig> from the atypical val166-ser <dig> loop makes one of the six hydrogen bonds present in the dimer interface while three other residues in the same loop contribute to van der waals interactions.
we suggest that the atypical loop found in 10093b may help support dimerization.
the absence of this loop in 10093f may explain the monomeric solution behavior of this family member.
structure and sequence relationships and homology modeling
in an effort to annotate the function of these two related proteins, bioinformatics analysis with the experimental structures was performed using dali  <cit> .
for 10093b, a dali search revealed only two structural matches with a z-score greater than  <dig>  the closest match, af <dig> from archaeoglobus fulgidus , a protein of unknown function belonging to pfam duf <dig> gave a z-score of  <dig>  with sequence identity of 15% and rmsd of  <dig>  Å between  <dig> structurally equivalent α-carbon atomic pairs.
thus, structure determination of 10093b not only provided the first structural information for the pfam family duf <dig>  it also documented unexpected structural similarity to a member of the duf <dig> pfam family that could not have been reliably predicted from sequence comparisons alone.
a dali search with 10093f revealed  significant matches  with af <dig> z-score  <dig> , as expected, and with a nitrate transport protein  z-score of  <dig>  with sequence identity of 12% and rmsd between  <dig> structurally equivalent α-carbon atomic pairs of  <dig>  Å. stereoviews of the superimposed polypeptide chains of 10093b over 10093f-monomers, -dimers and 10093b over 1zbm are presented in figure 3a, 3b and 3c respectively.
a blast  <cit>  search of uniprot protein sequence database using the sequences of both 10093b and 10093f yielded  <dig> matches .
virtually all of the matches are identified as bacterial or archaeal hypothetical proteins.
the three exceptions are as follows: a sam-dependent methyltransferase from lactococcus lactis , leucyl-trna synthetase from xanthomonas campestris , d-alanine-d-alanine ligase from nitrosomonas europaea .
at the time of publication, the experimental structures of 10093b and 10093f were used as a template to compute homology models of  <dig> proteins with related sequences with the modweb server  <cit> .
the  <dig> models out of  <dig> had sequence identity of great than 30%.
active site/ligand binding site prediction
active site/ligand binding site prediction performed using castp  <cit>  revealed two major clefts on the surfaces of the 10093b and 10093f .
further analysis of these surface features together with a multiple sequence alignment performed in clustalw  <cit>  and edited in bioedit  <cit>   performed for 10093b reveals the presence of most of the conserved residues, including asn <dig>  pro <dig>  ser <dig>  ser <dig>  ser <dig>  ile <dig>  gly <dig>  and asp <dig> .
we suggest that this larger cleft represents the active site and/or ligand binding site for this functionally uncharacterized pfam family.
prediction of putative function
10093b and 10093f belonging to duf <dig> family have remarkable similarity to duf <dig> protein family and a nitrate-binding protein .
this similarity was not evident from sequence comparison alone.
moreover, the uncharacterized duf <dig> family of proteins is annotated to be putative solute-binding protein.
our analysis of the surface clefts shows that the major cleft identified in 10093b is common to all of them, the size being nearly the same.
the binding pocket of 10093b superposes well with that of nitrate-binding protein.
further analysis of the active site shows that the entry of this cavity is occupied by hydrophobic residues as in 2g <dig>  <cit> .
in 10093b, the residual density in the difference fourier showed a dumb bell shaped density and was modeled as water.
this could very well be an unidentified ion.
based on the comparison of structures and binding sites, we predict that this protein could be a solute binding protein, though we cannot at this stage identify the nature of the ion.
the weak homology implies that it may be a different kind of solute.
CONCLUSIONS
the structure determination of 10093b and 10093f has revealed the unexpected similarity between duf <dig> and duf <dig> family of proteins suggesting that the information from three-dimensional structures along with the sequence information will predict the family of proteins of similar functions more reliably.
we have predicted the protein to be a solute-binding protein based on structure and binding cavity.
fold and structural similarity among proteins with low sequence identity  is not uncommon.
one prominent example would be proteins classified into different pfams but with the same tim barrel fold  <cit> .
amidohydrolases with low sequence homology have various substrate specificities and different enzymatic functions but all of them have similar fold.
these belong to different pfam families but are grouped together as a super family  <cit> .
this also suggests that duf <dig> and duf <dig>  though belonging to different pfams of unknown functions, may be members of the same superfamily.

 pfam is a comprehensive collection of protein domains and families, with a range of well-established information including genome annotation. each family in pfam is represented by two multiple sequence alignments and two profile-hidden markov models. pfam has two large series of functionally uncharacterized families, known as domains of unknown function and uncharacterized protein families. dufs are families that have been created by pfam whereas upfs are those created by swiss-prot and added to pfam. the protein structure initiative- has undertaken the task of structurally characterizing all pfam families that have no structural representation. the pfam protein family duf consists of proteins of unknown function, from bacteria and from archaebacteria. herein, we report the first crystal structures of duf family members, including q9rxe from deinococcus radiodurans and q9l0t streptomyces coelicolor and show that they are remarkably similar to pfam family duf  the structure of 10093b was determined to Å resolution using selenium-sad. the final refined model of 10093b contains protomers, sulfate ions, and water molecules. the final refined model of 10093f contains protomers and water molecules. despite low sequence identity, the polypeptide chain structures are very similar. the two structures superimpose well with a rmsd of Å for α-carbon atomic pairs, excluding a loop region between val to ser in 10093b that does not occur in 10093f. this region is absent in all other sequences shown in figure except in thermus thermophilus. values for the highest resolution shell are given within parentheses. 1rmerge = Σ|ii-⟨i⟩|/Σ|ii| where ii is the intensity of the ith measurement, and ⟨i⟩ is the mean intensity for that reflection. 2phasing power and 3fom as defined in sharp. 4rfactor = Σ||fobs|-|fcalc||/Σ|fobs| where |fcalc| and |fobs| are the calculated and observed structure factor amplitudes, respectively. notwithstanding the similarity of the two polypeptide chain folds, mr attempts with various 10093b-derived search models were not successful. molecular replacement may have failed because of low sequence identity and/or due to the presence of multiple protomers within the asymmetric unit. biologically active units 10093b eight monomers a, b, c, d, e, f, g, and h found in the asymmetric unit form four tightly packed dimers. each dimer pair superimposes very well on the remaining three with rmsds = Å– Å for all α-carbon atomic pairs. a dimer interface analysis performed using pdbsum demonstrated that each pair buries ~ Å of solvent accessible surface area, a value higher than would be found typically in interacting surfaces for a protein of this size. at least residues from each half of the dimer participate in intermolecular interactions. there are direct hydrogen bonded interactions between the protein atoms across the dimer interface. such a tight dimer interface suggests that the 10093b dimer is functional, which is supported by the results of analytical gel filtration. 10093f four monomers a, b, c, and d found in the crystallographic asymmetric unit occur as two dimers, albeit with an intermolecular packing arrangement that differs from that seen for 10093b. each observed 10093f dimer pair buries ~ Å of solvent accessible surface area, which is not thought to be significant for a protein of this size. moreover, the gel-filtration analysis revealed that 10093f is a monomer in solution. in the 10093b dimer, glu from the atypical val166-ser loop makes one of the six hydrogen bonds present in the dimer interface while three other residues in the same loop contribute to van der waals interactions. we suggest that the atypical loop found in 10093b may help support dimerization. the absence of this loop in 10093f may explain the monomeric solution behavior of this family member. structure and sequence relationships and homology modeling in an effort to annotate the function of these two related proteins, bioinformatics analysis with the experimental structures was performed using dali. for 10093b, a dali search revealed only two structural matches with a z-score greater than the closest match, af from archaeoglobus fulgidus, a protein of unknown function belonging to pfam duf gave a z-score of with sequence identity of 15% and rmsd of Å between structurally equivalent α-carbon atomic pairs. thus, structure determination of 10093b not only provided the first structural information for the pfam family duf it also documented unexpected structural similarity to a member of the duf pfam family that could not have been reliably predicted from sequence comparisons alone. a dali search with 10093f revealed significant matches with af z-score, as expected, and with a nitrate transport protein z-score of with sequence identity of 12% and rmsd between structurally equivalent α-carbon atomic pairs of Å. stereoviews of the superimposed polypeptide chains of 10093b over 10093f-monomers, -dimers and 10093b over 1zbm are presented in figure 3a, 3b and 3c respectively. a blast search of uniprot protein sequence database using the sequences of both 10093b and 10093f yielded matches. virtually all of the matches are identified as bacterial or archaeal hypothetical proteins. the three exceptions are as follows: a sam-dependent methyltransferase from lactococcus lactis, leucyl-trna synthetase from xanthomonas campestris, d-alanine-d-alanine ligase from nitrosomonas europaea. at the time of publication, the experimental structures of 10093b and 10093f were used as a template to compute homology models of proteins with related sequences with the modweb server. the models out of had sequence identity of great than 30%. active site/ligand binding site prediction active site/ligand binding site prediction performed using castp revealed two major clefts on the surfaces of the 10093b and 10093f. further analysis of these surface features together with a multiple sequence alignment performed in clustalw and edited in bioedit performed for 10093b reveals the presence of most of the conserved residues, including asn pro ser ser ser ile gly and asp. we suggest that this larger cleft represents the active site and/or ligand binding site for this functionally uncharacterized pfam family. prediction of putative function 10093b and 10093f belonging to duf family have remarkable similarity to duf protein family and a nitrate-binding protein. this similarity was not evident from sequence comparison alone. moreover, the uncharacterized duf family of proteins is annotated to be putative solute-binding protein. our analysis of the surface clefts shows that the major cleft identified in 10093b is common to all of them, the size being nearly the same. the binding pocket of 10093b superposes well with that of nitrate-binding protein. further analysis of the active site shows that the entry of this cavity is occupied by hydrophobic residues as in 2g. in 10093b, the residual density in the difference fourier showed a dumb bell shaped density and was modeled as water. this could very well be an unidentified ion. based on the comparison of structures and binding sites, we predict that this protein could be a solute binding protein, though we cannot at this stage identify the nature of the ion. the weak homology implies that it may be a different kind of solute.  the structure determination of 10093b and 10093f has revealed the unexpected similarity between duf and duf family of proteins suggesting that the information from three-dimensional structures along with the sequence information will predict the family of proteins of similar functions more reliably. we have predicted the protein to be a solute-binding protein based on structure and binding cavity. fold and structural similarity among proteins with low sequence identity is not uncommon. one prominent example would be proteins classified into different pfams but with the same tim barrel fold. amidohydrolases with low sequence homology have various substrate specificities and different enzymatic functions but all of them have similar fold. these belong to different pfam families but are grouped together as a super family. this also suggests that duf and duf though belonging to different pfams of unknown functions, may be members of the same superfamily. 
pfam is a comprehensive collection of protein domains and families, with a range of well-established information including genome annotation. unexpectedly, we found that duf pfam is remarkably similar to pfam family duf suggesting that the sequence-based classification alone may not be sufficient to classify proteins into pfam families. pfam has two large series of functionally uncharacterized families, known as domains of unknown function and uncharacterized protein families. crystal structures of two proteins from deinococcus radiodurans and streptomyces coelicolor belonging to pfam protein family duf have been determined using selenium-single-wavelength anomalous dispersion.
 pfam is a comprehensive collection of protein domains and families, with a range of well-established information including genome annotation. pfam has two large series of functionally uncharacterized families, known as domains of unknown function and uncharacterized protein families.  crystal structures of two proteins from deinococcus radiodurans and streptomyces coelicolor belonging to pfam protein family duf have been determined using selenium-single-wavelength anomalous dispersion. based on the structure, we have identified the putative function for this family of protein.  unexpectedly, we found that duf pfam is remarkably similar to pfam family duf suggesting that the sequence-based classification alone may not be sufficient to classify proteins into pfam families. 