dataesr/scientific-paragraphs-categorization huggingface dataset:
Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven variants, linked to heterogeneity in the assembly machineries. Here we focus on PilO2 Bp , a protein component of the Tfpb R64 thin pilus variant assembly machinery from the pathogen Burkholderia pseudomallei. PilO2 Bp belongs to the PF06864 Pfam family, for which an improved definition is presented based on newly derived Hidden Markov Model (HMM) profiles. The 3D structure of the Nterminal domain of PilO2 Bp (N-PilO2 Bp ), here reported, is the first structural representative of the PF06864 family. N-PilO2 Bp presents an actin-like ATPase fold that is shown to be present in BfpC, a different variant assembly protein; the new HMM profiles classify BfpC as a PF06864 member.
Among these, the Tfp7 locus was recognized as a putative operon/regulon containing nine genes coding for proteins BPSS1593 to BPSS1601. Tfp7 is of particular interest as it encodes for a Tfpb that is closely related to all members of the R64 thin-pilus variant. The operons of this variant share, along with Tfp7, genes encoding proteins belonging to the same Pfam  [19]  families, and particularly a specific protein, BPSS1599, of unknown function. Pfam analysis of BPSS1599, a predicted accessory Pilus assembly protein 2 (PilO2 Bp ) revealed an inconsistency between the HMM profile of the PF06864 Pfam family and the proposed PilO2 Bp sequence alignment. Based on sequence searches, we present three new HMM profiles that allow a full alignment of PilO2 Bp with its Pfam family. Moreover, we report the 1.
55 A crystal structure of the N-terminal domain of PilO2 Bp (residues 1-192; N-PilO2 Bp ), solved by S-SAD phasing, providing a first glimpse of the 3D structural properties of the PF06864 family, previously lacking a structure representative. We show that N-PilO2 Bp consists of two a/b sub-domains separated by a prominent central cleft, typical of actin-like ATPases. Additionally, in line with the description of PilO2 Bp as a putative Tfpb assembly machinery protein, N-PilO2 Bp is found to be homologous to N-BfpC, another cytoplasmic protein involved in a Tfpb assembly system. Interestingly, BfpC is a specific protein of the Bundleforming pilus variant, which differs from the R64 thin-pilus variant to which PilO2 Bp belongs. Both structures were found to be very similar, despite lack of any significant sequence identity, and despite the fact that BfpC had not been initially recognized as PF06864 member.
Cloning, purification and crystallization The 59 end of the BPSS1599 gene (NCBI accession number YP_111607. 1), coding for N-PilO2 Bp , amino acids 1-192, was amplified by PCR from genomic DNA (Prof. Titball's group, University of Exeter, UK) from B. pseudomallei strain K96243 using the primers PilO2-F1 (59-CACCATGAGCGCGCAGGTG-39) and PilO2-R1 (59-CTACGACAGACGCCGCTCG-39) for insertion into the pET151/TOPO vector (Life Technologies). The same protocol was applied to the 39 end of the BPSS1599 gene, coding for amino acids 221 to 432. Successful cloning and PCR fidelity were confirmed by sequencing (BMR Genomics Srl. , Padova). N-PilO2 Bp and C-PilO2 Bp domains were expressed as Nterminal His-tag fusion proteins in C41 (DE3) E. coli cells in Luria-Bertani broth, inducing with 0. 5 mM IPTG at 18uC overnight.
Generation and validation of the HMM profiles for Pfam family PF06864 A sequential strategy was applied to improve the HMM profiles for the PF06864 family. First, the full sequences of the PF06864 RP15 group members, including PilO2 Bp , were realigned against the original PF06864 HMM profile using the 'hmmalign' tool from the HMMR3 package 20 (Alignment 3). Residues comprising alignment positions 1 to 170 in every sequence were then extracted from Alignment 3 for independent alignment. Alignment position 171 contains PilO2 Bp Ala93, the first residue aligned with the PF06864 profile. The isolated N-terminal sequences were then used as input for multiple alignment with 'T-Coffee'  [20] , run in three modes: default parameters (Alignment 4A), accurate mode using the EBI psi-blast client (Alignment 4B) and accurate mode using the NCBI blastp client (Alignment 4C).
Alignments 4A, B and C were merged with Alignment 3 by substitution of the first 170 residue positions in the latter, thus generating three new alignments, with complete sequences, for the PF06864 RP15 group (Alignments 5A-C). These alignments were used to generate three new HMM profiles (Default HMM profile, EBI HMM profile and NCBI HMM profile) with the 'hmmbuild' tool of HMMR3. In order to validate the new HMM profiles, the original PF06864 seed sequences were aligned against them, obtaining three new multiple alignments (Alignments 8A-C). Alignments 8A-C were then compared to the PF06864 seed multiple alignment using the T-Coffee 'profile vs profile' function, resulting in a score of 98 (out of 100) using the Default and NCBI HMM profiles (Alignments 9 A, B and C, respectively) and a score of 97 using the EBI HMM profile (Alignment 9B).
As a reference, a score of 99 is obtained when comparing Pfam's PF06864 seed multiple alignment against itself (Alignment 9D). Finally, the alignment of PilO2 Bp and of the rest of the PF06864 RP15 group members (Alignments 6A-C) with the new profiles was analyzed. As expected, the new profiles now cover all the sequence, in contrast to Alignment 1, which excludes the first 92 residues.
X-ray diffraction Data Collection, Structure Determination and Refinement Successful S-SAD phasing often relies on the presence of additional/unexpected weak anomalous scattering species (a phosphate ion in our case) in addition to the protein Met/Cys sulphur atoms. The method requires a long wavelength for the incident X-ray beam in order to maximize the f '' anomalous contribution of the S atoms. When using long wavelength (lower X-ray energy) for data collection, the harmonic contaminations of the X-rays affect the anomalous signal. Suppression or reduction of higher harmonic contamination in the primary X-ray beam is an essential precondition for success of an S-SAD experiment. All diffraction datasets for N-PilO2 Bp (containing phosphate) were collected on the BM14 beam line, at the European Synchrotron Radiation Facility (ESRF, France) using a MAR 225 CCD detector on a single good quality tetragonal crystal (Space group P4 1 2 1 2, unit cell edges a = b = 56.
0 A ˚; c = 117. 0 A ˚; a = b = c = 90. 0u). The 1. 55 A ˚resolution data set was collected at beam energy of 12. 7 keV, and treated as a native dataset. The sulfur-SAD data sets were collected at 7 keV (l = 1. 7712 A ˚) by exploiting the goniostat k geometry (k = 0u, k = 35u and k = 70u), to limit systematic errors associated with X-ray absorption or radiation damage, and to achieve high multiplicity within the collected data. The harmonic contamination from 21 keV (for the 7 keV set) was reduced by offsetting the second crystal of the beam monochromator using the pusher value -0. 25. Datasets were integrated with the program HKL2000, and scaled with SCALEPACK  [21]. Data collection statistics are shown in Table  1. Attempts at S-SAD phasing were successful when employing the merged k = 0u and k = 35u S-SAD datasets, measured to 1.
9 A ˚resolution. The ShelxC/D/E programs embedded in the HKL2MAP application was used to analyze the heavy-atom substructure of the datasets (ShelxC), locate the anomalous scatterers (ShelxD), and extract phase information (ShelxE)  [22]. About 100 trial runs of ShelxD were performed to find the correct positions of the anomalous scatterers. All four expected S atoms were located along with an additional peak for a phosphor atom. The 'heavy atoms' were subjected to 20 cycles of phase refinement in ShelxE, and three cycles of model tracing, while extending data resolution, using free lunch algorithm in ShelxE. The experimental phases and the linked model were employed for automated model building in Phenix -AutoBuild program  [23] [24] [25] [26] [27] , which allowed to build a model consisting of 194 residues, with an overall model/map correlation coefficient of 0.
884. Inspection of the map confirmed the presence of one phosphate ion, and of two additional residues at the N-terminal (residues belonging to the vector used). Several rounds of manual model building with COOT, and refinement with the program REFMAC5, were carried over  [26, 28, 29]. The structure was refined to R cryst = 0. 185, R free = 0. 223 values, and the quality of the model checked with PROCHECK  [30]. The final refinement statistics and quality parameters are shown in Table  2. The diffraction dataset, for PilO2 Bp devoid of phosphate, was collected on ID23-1 beam line, at the European Synchrotron Facility (ESRF, France), on a single good quality tetragonal crystal (Space group P4 1 2 1 2, unit cell edges a = b = 52. 7 A ˚; c = 127. 0 A ˚; a = b = c = 90. 0u). The data were integrated with the program iMOSFLM and scaled with SCALA; data collection statistics are reported in Table  1.
Phases were obtained using the N-PilO2 Bp bound to phosphate structure by molecular replacement. The structure was further completed manually and refined by cycling between COOT and REFMAC5 programs, refined to R cryst = 0. 194, R free = 0. 250, and the quality of the model checked with PROCHECK. The final refinement statistics and quality parameters are shown in Table  1. The atomic coordinates and structure factors for N-PilO2 Bp with phosphate, and without the bound phosphate, were deposited in the RCSB Protein Data Bank under accession codes 4BYZ and 4BZ0, respectively  [31].
Results PilO2 Bp is a component of the Tfpb R64 thin pilus variant PilO2 Bp is described as belonging to the PF06864 Pfam family, which consists of several enterobacterial specific PilO proteins; PilO2 Bp is annotated as a specific structural part of a Tfpb assembly apparatus. Notably, two PilO Pfam families have been described and documented in a recent review  [32]. The first PilO family, PF04350, hosts proteins of the Tfpa assembly machinery (no homologs to members of this family have been detected in B. pseudomallei K96243). The second PilO family, PF06864, includes proteins involved in the Tfpb R64 thin pilus variant, such as PilO2 Bp. To ease nomenclature, in the following we will address the PF06864 family as PilO2, and add the suffix 2 to all components of the Tfpb R64 thin pilus variant (Pil).
In support of the Pfam family description, we noted that, according to the Burkholderia Genome Database  [33] , PilO2 Bp pertains to a predicted operon (positioned from 2167547 to 2177170 bp on chromosome 2 of B. pseudomallei K96243), composed of nine open reading frames coding for putative Pil proteins (PilV2, M2, S2, R2, Q2, P2, O2, N2) and for the TcpQ2 protein, with an estimated length of 9623 bp. Downstream to this operon, a pilT2 gene codes for the PilT2 protein, an additional component of the Tfp7 assembly machinery (Fig. 1 ). The organization of Tfp7 is well conserved in all Burkholderia strains, suggesting a very ancient origin for this genetic element. Although the genetic organization is not conserved among the Tfpb R64 thin pilus variant operons, all genes are orthologs (Fig. 1 ).
PilO2 Bp within the PF06864 Pfam family As already mentioned, based on the Pfam sequence-search tool PilO2 Bp (UniProt  [37]  code Q63JW5) was assigned to family PF06864. In fact, PilO2 Bp is part of the alignment of the proteins from the 15% representative proteomes (RP15)  [38]  for this family (Alignment 1). However, alignment of PilO2 Bp to the PF06864 profile begins at Ala93, thus excluding the first 92 residues and casting doubts on whether PilO2 Bp is correctly assigned only as a PilO2 protein. Pfam reports residues 12 to 92 as belonging to the 'envelope', however they are out of the alignment and a huge gap is introduced in their place. After careful examination of the PF06864 RP15 it was possible to manually align a short motif in the N-terminal region of these proteins (Alignment 2).
This suggested that the first 92 PilO2 Bp residues may in fact belong to the PilO2 profile. In order to confirm this hypothesis, a sequential strategy, schematized in Fig. 2 , was applied. To see whether the first positions in the family profile may be improved, new HMM profiles were generated. To this end, the program T-Coffee  [20]  was run in three different modes: default parameters, accurate mode using the EBI psi-blast client, and accurate mode using the NCBI blastp client. The three new HMM profiles and all generated alignments are available for download at http:// bioinf. uab. cat/newPF06864hmmprof/. Three complete alignments of the full-length protein, which we shall call Default HMM profile, EBI HMM profile and NCBI HMM profile, respectively, were then obtained and validated for PilO2 Bp (Alignments 10 A, B and C).
Updated HMM profiles identify new members of the PF06864 family The PF06864 members are described as pilin accessory protein (PAP_PilO) in the Pfam database. The family is composed of 257 protein members, with proteins A3JI41 and Q6EVW5 (UniProt Id) having the lowest and highest scores, respectively. Six of these proteins have been however removed from UniProt, therefore reducing the actual set to 251 members. In search for new members of PF06864, we first scanned the whole uniprot_ trembl_bacteria database (http://www. uniprot. org) with the original Pfam HMM profile. Using the hmmsearch program from HMMR3  [39] , 391 matches were found with significance above the default threshold, including the 251 proteins reported in Pfam for PF06864 and with A3JI41 as the member with lowest score. All 391 sequences are annotated in UniProt as belonging to the PF06864 family.
Resolution range (A ˚) 50-1. 9 (1. 9- We then scanned the uniprot_trembl_bacteria database using our three new HMM profiles. Out of the 251 PF06864 members, 250 scored over the significance threshold. The missing sequence (UniProt Id G1ZDN7) yielded also a positive match when manually aligned against the profiles using the hmmalign program from HMMR3 (204 of the 246 residues in the sequence align using the original Pfam profile, while 184 residues align based on the new Default and NCBI HMM profiles, and 183 with the EBI profile). In addition, and notably, one hmmsearch identified 182 supplementary sequences matching the EBI HMM profile, two of which (G4FYD9 and K2RGC3) have been recently removed from UniProt, and 179 sequences matching the Default and NCBI HMM profiles. The latter 179 sequences are fully contained in the EBI-profile-matching set of 180.
Out of the 180 additional proteins, 137 are already annotated in UniProt as members of the PF06864 family. Of the remaining 43 proteins, eight are annotated as PilO or pili-related proteins, two as ATPase, two as BfpC, and 31 as putative or uncharacterized proteins (Table  S1 ). A search in the Swiss-Prot database for proteins matching the three new profiles showed that the majority of matches belong to plant or animal bacterial pathogens, with the remainder being symbionts. Remarkably, similar results were obtained for the 43 newly-identified putative members of the family.
The tetragonal crystals, grown from sodium/potassium phosphate solutions at pH 7-8, proved of excellent diffraction quality (see Materials and Methods and Table  1 ). Due to the lack of suitable structure homologs in the protein data bank (PDB), the use of molecular replacement to solve N-PilO2 Bp 3D structure was prevented. Thus, considering the availability of 1. 5 A ˚resolution data for this protein, the singlewavelength anomalous diffraction (SAD) phasing method was adopted based on the four intrinsic sulphur atoms present in native N-PliO2 Bp. S-SAD data collection and phasing was conducted at the BM14 diffraction beam line at the ESRF (Grenoble, France), using 1. 77 A ˚X-ray source wavelength. Four sulphur and one phosphor anomalous scatterers were located during the phasing procedure.
In full-length PliO2 Bp , such a pocket may face the membrane and fall in its close proximity, thus promoting the interaction with phospholipids. In the crystal, the phosphate ion further interacts with Arg50 and Arg77 of a symmetry-related molecule. N-PilO2 Bp and the Tfpb bitopic protein N-BfpC share the same fold As N-PilO2 Bp had no evident structural homologs known, we used our crystallographic results to search the structural database. Using Dali  [44] , the closest structural homolog of N-PilO2 Bp was identified as the N-terminal domain of BfpC (Dali Z-score: 16. 8, PDB Id 3VHJ, root-mean-square difference (RMSD) of 2. 9 A ˚over 159 matched Ca pairs; hereafter N-BfpC), an accessory protein of the E. coli Tfpb BFP variant. As highlighted by the low RMSD value, the two structures are very similar, but differ for the absence of the anchor-loop in N-BfpC (residues 168-179 in N-PilO2 Bp , Fig.
3F ); nevertheless, the two proteins are described as part of two distinct assembly machineries. The next Dali hit corresponds to an uncharacterized protein from Bacteroides thetaiotaomicron (PDB 3HRG) with an actin-like ATPase fold (Z-score = 7. 4). Based on amino-acid sequence only, BfpC is not recognized as a member of a Pfam family, a result that would stress substantial evolutionary distance from PilO2 Bp. However, a PDB search with the recently introduced PDBfam tool  [45]  recognizes 3VHJ as the only PDB structure matching the PF06864 Pfam family. Three additional results support BfpC as a member of PF06864. First, alignment of the full BfpC sequence (B7UTD4) against our new profiles resulted in 69% (Default HMM profile), 62% (EBI profile) and 64% (NCBI profile) of the residues aligned.
Second, using the function READALIGN of the ProFit program (http://www. bioinf. org. uk/software/profit/index. html) a superposition of the 3VHJ structure onto N-PilO2 Bp , based on the sequence alignment produced by our HMM profiles produced low RMSD values for the aligned part (3. 6 A ˚using the Default profile, 3. 2 A ˚using the EBI profile and 2. 8 A ˚using the NCBI profile). Thirdly, a search of PilO2 Bp against uniprot_trembl_bacteria with the jackhmmer program (HMMR3 package) using default parameters (five iterations)  [46]  (Pfam main protocol for generating families  [19] ) returned a multiple alignment where both proteins are present. Using this alignment to superpose both structures results in an RMSD of 3. 0 A ˚. The three pairwise alignments mentioned above, together with a structural alignment obtained with the program CE  [47]  are presented in Fig.
pseudomallei. Despite the fact that PilO2 Bp , is a representative member of the PF06864 Pfam family, the Pfam algorithm failed to align the first 92 residues of this protein with the other PF06864 members. Pfam is a widely used database of protein families, currently containing more than 13000 manually curated families. Two types of families are distinguished: high quality, manually curated Pfam-A families, and automatically generated Pfam-B families. Some Pfam-A familes are seeded by structures deposited in the Protein Data Bank, and the determination of new structures for known families has already led to their extension in the past  [19, 49]. Extensive sequence analysis within the 15% representative PF06864 members indicated that the definition of PF06864 is incomplete. Indeed, the alignment of the N-terminal part of these proteins could be substantially improved using new HMM profiles for the family, as demonstrated here.
In addition, screening the uniprot_trembl_bacteria database with our newly developed profiles allowed us to identify 43 new PF06864 family members that are likely to be PilO2 proteins. Prior to the results reported here, the PF06864 family did not have a representative 3D structure. Our SAD crystallographic approach, based on anomalous scattering from sulfur atoms, yielded a high resolution N-PilO2 Bp 3D structure, thus shedding first light on the key structural features of this protein family. The N-PilO2 Bp 3D structure, according to SCOP, hosts a ribonuclease H-like fold, typical of proteins belonging to the actin-like ATPase domain superfamily, consisting of a globular moiety composed of two similar a/b sub-domains separated by a cleft. One of the N-PilO2 Bp crystal packing interfaces hosts a phosphate ion, housed in a pocket that could be functionally relevant in vivo, mediating the binding to phospholipids of the IM.
On the other hand, the stabilization of the (otherwise flexible) 'anchor loop' built by residues 169-179 is obtained thanks to intermolecular interactions that occur in a region characterized by positively charged residues, suggesting its potential role in the assembly with other (macro)molecular partners. N-BfpC is the closest known structural homolog of N-PilO2 Bp (RMSD of 2. 9 A ˚). Although BfpC is a Tfpb system component, its assembly machinery pertains to a variant different from Tfp7. However, the two operons share four orthologous genes, with bfpC and pilO2 Bp being non-orthologous (sequence identity of 6. 7%; sequence similarity of 16. 7%). In fact, BfpC had not been assigned to the PF06864 family. Its belonging to this family becomes however clear when the HMM profiles described here are applied.
Notably, N-BfpC was identified as a structural homolog of N-EpsL from the Type II secretion system  [51]. N-EpsL interacts with the N-terminal part of the EpsE ATPase. The PilT2 Bp ATPase from Tfp7 shares 19% homology with EpsE, but lacks the first 110 residues that are responsible for this association. Such structural features may imply that PilT2 Bp and PilO2 Bp do not associate, or that their mutual recognition is based on different principles. In conclusion, coupling thorough sequence analyses, database mining, and a new S-SAD phased crystal structure, led to two innovative discoveries within the PF06864 Pfam family. On one hand, the establishment of new HMM profiles enabled a full sequence alignment of PilO2 Bp to other members of the family and prompted the identification of 43 new members.
The third dimer is formed by the green and blue monomers. D. Stereo view of the electron density map for the residues building the phosphate ion binding pocket. The phosphate ion is shown as sphere; the electron density is contoured at 1. 5 sigma level. E. Front and back view of N-PilO2 Bp electrostatic surface potential. The electrostatic potential was calculated using the CCP4MG viewer. Negative (red) and positive (blue) charges, and uncharged (white) surfaces are shown. F. Superposition of the 3D structures of N-PilO2 Bp (cyan; PDB codes 4BYZ and 4BZ0) and N-BfpC (chocolate; PDB code 3VHJ). doi:10. 1371/journal. pone. 0094981. g003.
Acknowledgements We wish to thank  Roger Lala Andriamiarisoa  for the fine illustrations, and the flora team of  Rio Tinto-QMM  ( Johny Rabenantoandro ,  Faly Randriatafika ,  David Rabehevitra  and  Roger Ramison ) for providing excellent collections and assistance in the field in SE Madagascar. We are also very grateful for the valuable comments on the manuscript provided by  Sven Buerki ,  Martin Callmander  and an anonymous reviewer. LN is grateful to  Laurent Gautier ,  Rodolphe Spichiger  and  Pierre-André Loizeau  for support,  Patrick Ranirison  for providing herbarium specimens, and  Felipe Castaño  for assistance with the preparation of material for microscopic observation. Fieldwork by MBG botanists was conducted under collaborative agreements between the  Missouri Botanical Garden  and the  Parc Botanique et Zoologique de Tsimbazaza  and the  Direction de la Recherche Forestière et Piscicole, FOFIFA, Antananarivo, Madagascar.