L Laboratory, Upton, New York) using a Quantum 4-CCD detector (Area

L Laboratory, Upton, New York) using a Quantum 4-CCD detector (Area Detector Systems Corp Poway, CA, USA) and processed using HKL2000 [24]. Heavy atom (Se) location, phasing and density modification were performed using the program ShelxC/D/E [25], and model building was carried out with the program Buccaneer [26] in CCP4. When necessary, the model was manually built in COOT [27] and refinement was performed in Refmac5 [28]. At the final stage of refinement, well-ordered water molecules were included. The model had good Lixisenatide stereochemistry, as analyzed by PROCHECK [29] (Table 1). All structure-related figures reported in this manuscript were prepared using PyMol [30].28.58?.00 0.20 (21561) 0.23 (1191) 0.014 1.2208 1034.25 39.86 46.92.2 7.0 0.8 0.Protein Data Bank Accession CodeCoordinates and structure factors of the ligand-free Ca2+/Zn2+/ CaM has been deposited with RCSB Protein Data Bank with codes 4HEX.Rsym = g|Ii ?,I.|/|Ii| where Ii is the intensity of the ith measurement, and ,I. is the mean intensity for that reflection. b Reflections with I.s was used in the refinement. c Rwork = |Fobs ?Fcalc|/|Fobs| where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively. d Rfree = as for Rwork, but for 5? of the total reflections chosen at random and omitted from refinement. e Individual B-factor refinements were calculated. *The high buy PHCCC resolution bin details are in the parenthesis. doi:10.1371/journal.pone.0054834.tResults and Discussion Overall StructureThe crystal structure of ligand-free CaM was determined by Single-wavelength Anomalous Dispersion (SAD) method and ?refined to an R-factor of 0.20 (Rfree = 0.23) up to 2.02 A resolution. The electron density for the N-terminal His-tag and the first 5 residues were not well-defined and were therefore notincluded in the model. The model had good geometry, with no residues in the disallowed region of the Ramachandran plot ?(Table 1). There are two identical molecules (RMSD 0.7 A) in the asymmetric unit (Supplementary Figure S1). However, the PISA server [31] analysis and gel filtration chromatography suggest that ligand-free CaM is a monomer. The Ca2+ ions were well-defined in the electron density map, located at each EF-hand motif. Besides a Zn2+ ion was observed near His108 for the first time in CaM. The ligand-free CaM molecule was in an open conforma-A Novel Conformation of CalmodulinFigure 1. A novel trans conformation of calmodulin (CaM). A: Ribbon representation of novel conformation of ligand-free CaM. The helices, loops, Ca2+ and Zn2+ are shown in blue, pale green, green and grey, respectively. The CaM molecule adopts an extended dumbbell-shaped conformation and the two domains are well separated. The bending at Arg75 is shown in stick representation. The a-helices are numbered from I-VIII. B: 2Fo-Fc electron density map for the region 65?0 aa of CaM. This map 24786787 is contoured at a level of 1317923 1s. doi:10.1371/journal.pone.0054834.gtion, wherein helices of EF motifs were perpendicular to each other (Figure 1).Calmodulin Adopts a Novel ConformationA search for structural homologs in the PDB database using the DALI program [32] did not identify any similar structures. In the present structure, the ligand-free CaM adopts a unique conformation, with the relative disposition of the two lobes being completely different to any of the previously reported CaM structures (Figure 2). However, a one-to-one comparison of individual domains showed no significant structural di.L Laboratory, Upton, New York) using a Quantum 4-CCD detector (Area Detector Systems Corp Poway, CA, USA) and processed using HKL2000 [24]. Heavy atom (Se) location, phasing and density modification were performed using the program ShelxC/D/E [25], and model building was carried out with the program Buccaneer [26] in CCP4. When necessary, the model was manually built in COOT [27] and refinement was performed in Refmac5 [28]. At the final stage of refinement, well-ordered water molecules were included. The model had good stereochemistry, as analyzed by PROCHECK [29] (Table 1). All structure-related figures reported in this manuscript were prepared using PyMol [30].28.58?.00 0.20 (21561) 0.23 (1191) 0.014 1.2208 1034.25 39.86 46.92.2 7.0 0.8 0.Protein Data Bank Accession CodeCoordinates and structure factors of the ligand-free Ca2+/Zn2+/ CaM has been deposited with RCSB Protein Data Bank with codes 4HEX.Rsym = g|Ii ?,I.|/|Ii| where Ii is the intensity of the ith measurement, and ,I. is the mean intensity for that reflection. b Reflections with I.s was used in the refinement. c Rwork = |Fobs ?Fcalc|/|Fobs| where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively. d Rfree = as for Rwork, but for 5? of the total reflections chosen at random and omitted from refinement. e Individual B-factor refinements were calculated. *The high resolution bin details are in the parenthesis. doi:10.1371/journal.pone.0054834.tResults and Discussion Overall StructureThe crystal structure of ligand-free CaM was determined by Single-wavelength Anomalous Dispersion (SAD) method and ?refined to an R-factor of 0.20 (Rfree = 0.23) up to 2.02 A resolution. The electron density for the N-terminal His-tag and the first 5 residues were not well-defined and were therefore notincluded in the model. The model had good geometry, with no residues in the disallowed region of the Ramachandran plot ?(Table 1). There are two identical molecules (RMSD 0.7 A) in the asymmetric unit (Supplementary Figure S1). However, the PISA server [31] analysis and gel filtration chromatography suggest that ligand-free CaM is a monomer. The Ca2+ ions were well-defined in the electron density map, located at each EF-hand motif. Besides a Zn2+ ion was observed near His108 for the first time in CaM. The ligand-free CaM molecule was in an open conforma-A Novel Conformation of CalmodulinFigure 1. A novel trans conformation of calmodulin (CaM). A: Ribbon representation of novel conformation of ligand-free CaM. The helices, loops, Ca2+ and Zn2+ are shown in blue, pale green, green and grey, respectively. The CaM molecule adopts an extended dumbbell-shaped conformation and the two domains are well separated. The bending at Arg75 is shown in stick representation. The a-helices are numbered from I-VIII. B: 2Fo-Fc electron density map for the region 65?0 aa of CaM. This map 24786787 is contoured at a level of 1317923 1s. doi:10.1371/journal.pone.0054834.gtion, wherein helices of EF motifs were perpendicular to each other (Figure 1).Calmodulin Adopts a Novel ConformationA search for structural homologs in the PDB database using the DALI program [32] did not identify any similar structures. In the present structure, the ligand-free CaM adopts a unique conformation, with the relative disposition of the two lobes being completely different to any of the previously reported CaM structures (Figure 2). However, a one-to-one comparison of individual domains showed no significant structural di.