Building were performed using AutoRickshaw and ArpWarp [44] and resulted in a readily interpretable map. For refinement, 5 of the reflections were removed for the calculation of Rfree. The model was further built using rounds of manual building in COOT [45] and refinement using phenix.refine [46]. The first refinement step included simulated annealing starting at 5000 K. The native structure was solved with MOLREP [47] using the FimP SeMet structure as the search model. Due to movements of the N-domain, only residues 190?90 were placed in the molecular replacement solution and residues 35?89 were built manually. The native structure was refined as the SeMet structure. The quality of the model was analyzed with WHATCHECK [48]. Ramachandran statistics were obtained 16985061 using COOT [45]. Crystallographic statistics are Licochalcone A cost presented in Table 1. The X-ray coordinates and structure purchase 58-49-1 factors have been deposited in the Protein Data Bank under accession codes 3UXF.Limited Proteolysis and N-terminal Sequencing of FimA and FimP FragmentsPurified recombinant FimP (A. oris strain T14V) and FimA (A. naeslundii strain 12104) in 20 mM CaCl2, 50 mM Tris-HCl pH 8.0, 1 glycerol and 2 mM DTT were incubated with trypsin at a ratio 1:100 trypsin: FimP/FimA at 293 K for 1 h. The cleavage products were separated on by SDS-PAGE and transferred to a PVDF membrane. N-terminal sequencing of the cleavage products was performed.Sequencing of FimP and FimA from A. oris and A. naeslundii StrainsThe 1313429 fimP genes were sequenced from A. oris clinical isolates (n = 42) and reference strains (n = 6; T14V, PK1259, P-1-N, P-8L, LY7 and P-1-K). The fimP genes were amplified by PCR using purified genomic DNA and the Ready-To-Go PCR polymerase kit (GE healthcare). The fimA genes were sequenced from A. oris (n = 14) and A. naeslundii (n = 17) isolates. The fimA genes were amplified by PCR from purified DNA using the Iproof High-Fidelity enzyme (BioRad). Purified fragments were cloned into Zero BluntH TOPOH PCR Cloning Kit (Invitrogen) and sequenced. All sequencing was performed by Eurofins MWG Operon. The fimP (corresponding to amino acids 31?16) and fimA (corresponding toFimP Structure and Sequence AnalysesFigure 7. Superposition of FimP and FimA. The FimP crystal structure superimposed onto the corresponding structure of FimA (M- and C domains). The FimP structure is depicted in light blue and FimA in green. The proline-rich segment in FimA is coloured in orange with the prolines as stick models. The equivalent loop in FimP is colored in purple. The metal ion coordinated by a FimP loop is depicted in grey. The N-terminal domain of FimP is colored in light grey. The figure is shown in stereo. doi:10.1371/journal.pone.0048364.gthe full length protein) sequences were assembled and analyzed using the CodonCode Aligner and MEGA5 software. Identity/ similarity values were generated using the NCBI web site.Author ContributionsConceived and designed the experiments: KP NS. Performed the experiments: KP RC AE. Analyzed the data: KP AE RC NS. Contributed reagents/materials/analysis tools: KP NS. Wrote the paper: KP NS.AcknowledgmentsWe are grateful for access to the beamlines ID23-1 and ID14-1, ESRF, Grenoble and for the support of the beamline staff.
The vertebrate MAP1 family of microtubule-associated proteins consists of three members, MAP1A, MAP1B, and MAP1S. MAP1A and MAP1B are .300 kDA proteins and are expressed at high levels in the central and peripheral nervous system in the adult and d.Building were performed using AutoRickshaw and ArpWarp [44] and resulted in a readily interpretable map. For refinement, 5 of the reflections were removed for the calculation of Rfree. The model was further built using rounds of manual building in COOT [45] and refinement using phenix.refine [46]. The first refinement step included simulated annealing starting at 5000 K. The native structure was solved with MOLREP [47] using the FimP SeMet structure as the search model. Due to movements of the N-domain, only residues 190?90 were placed in the molecular replacement solution and residues 35?89 were built manually. The native structure was refined as the SeMet structure. The quality of the model was analyzed with WHATCHECK [48]. Ramachandran statistics were obtained 16985061 using COOT [45]. Crystallographic statistics are presented in Table 1. The X-ray coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 3UXF.Limited Proteolysis and N-terminal Sequencing of FimA and FimP FragmentsPurified recombinant FimP (A. oris strain T14V) and FimA (A. naeslundii strain 12104) in 20 mM CaCl2, 50 mM Tris-HCl pH 8.0, 1 glycerol and 2 mM DTT were incubated with trypsin at a ratio 1:100 trypsin: FimP/FimA at 293 K for 1 h. The cleavage products were separated on by SDS-PAGE and transferred to a PVDF membrane. N-terminal sequencing of the cleavage products was performed.Sequencing of FimP and FimA from A. oris and A. naeslundii StrainsThe 1313429 fimP genes were sequenced from A. oris clinical isolates (n = 42) and reference strains (n = 6; T14V, PK1259, P-1-N, P-8L, LY7 and P-1-K). The fimP genes were amplified by PCR using purified genomic DNA and the Ready-To-Go PCR polymerase kit (GE healthcare). The fimA genes were sequenced from A. oris (n = 14) and A. naeslundii (n = 17) isolates. The fimA genes were amplified by PCR from purified DNA using the Iproof High-Fidelity enzyme (BioRad). Purified fragments were cloned into Zero BluntH TOPOH PCR Cloning Kit (Invitrogen) and sequenced. All sequencing was performed by Eurofins MWG Operon. The fimP (corresponding to amino acids 31?16) and fimA (corresponding toFimP Structure and Sequence AnalysesFigure 7. Superposition of FimP and FimA. The FimP crystal structure superimposed onto the corresponding structure of FimA (M- and C domains). The FimP structure is depicted in light blue and FimA in green. The proline-rich segment in FimA is coloured in orange with the prolines as stick models. The equivalent loop in FimP is colored in purple. The metal ion coordinated by a FimP loop is depicted in grey. The N-terminal domain of FimP is colored in light grey. The figure is shown in stereo. doi:10.1371/journal.pone.0048364.gthe full length protein) sequences were assembled and analyzed using the CodonCode Aligner and MEGA5 software. Identity/ similarity values were generated using the NCBI web site.Author ContributionsConceived and designed the experiments: KP NS. Performed the experiments: KP RC AE. Analyzed the data: KP AE RC NS. Contributed reagents/materials/analysis tools: KP NS. Wrote the paper: KP NS.AcknowledgmentsWe are grateful for access to the beamlines ID23-1 and ID14-1, ESRF, Grenoble and for the support of the beamline staff.
The vertebrate MAP1 family of microtubule-associated proteins consists of three members, MAP1A, MAP1B, and MAP1S. MAP1A and MAP1B are .300 kDA proteins and are expressed at high levels in the central and peripheral nervous system in the adult and d.
