A graphical user interface to the<i>CCP</i>4 program suite

Potterton, E. A.; Briggs, Peter; Turkenburg, M.G.W.; Dodson, E.J.

doi:10.1107/s0907444903008126

Cited by 1,208 publications

(962 citation statements)

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“…Data was reduced using HKL2000/SCALEPACK 45 or XDS/XSCALE. 46 Each structure was solved by sequential molecular replacement searches with PHASER 47 using Fab constant and variable regions (Protein Data Bank accession 4I77) as individual search models. Iterative rounds of model adjustment with COOT 48 were followed by simulated annealing, coordinate, and b-factor refinement with PHENIX 49 and BUSTER (Global Phasing Ltd) were performed to build the final models.…”

Section: Methodsmentioning

confidence: 99%

Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

Fong¹,

Kajihara²,

Chen³

et al. 2018

mAbs

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Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing health threat worldwide. Efforts to identify novel antibodies that target S. aureus cell surface antigens are a promising direction in the development of antibiotics that can halt MRSA infection. We biochemically and structurally characterized three patient-derived MRSA-targeting antibodies that bind to wall teichoic acid (WTA), which is a polyanionic surface glycopolymer. In S. aureus, WTA exists in both α- and β-forms, based on the stereochemistry of attachment of a N-acetylglucosamine residue to the repeating phosphoribitol sugar unit. We identified a panel of antibodies cloned from human patients that specifically recognize the α or β form of WTA, and can bind with high affinity to pathogenic wild-type strains of S. aureus bacteria. To investigate how the β-WTA specific antibodies interact with their target epitope, we determined the X-ray crystal structures of the three β-WTA specific antibodies, 4462, 4497, and 6078 (Protein Data Bank IDs 6DWI, 6DWA, and 6DW2, respectively), bound to a synthetic WTA epitope. These structures reveal that all three of these antibodies, while utilizing distinct antibody complementarity-determining region sequences and conformations to interact with β-WTA, fulfill two recognition principles: binding to the β-GlcNAc pyranose core and triangulation of WTA phosphate residues with polar contacts. These studies reveal the molecular basis for targeting a unique S. aureus cell surface epitope and highlight the power of human patient-based antibody discovery techniques for finding novel pathogen-targeting therapeutics.

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Section: Methodsmentioning

confidence: 99%

Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

Fong¹,

Kajihara²,

Chen³

et al. 2018

mAbs

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“…Using the HKL2MAP graphics user interface (T. Pape and T. Schneider, personal communication), native and putative derivative data sets were scaled together and analyzed with SHELXC (G. M. Sheld-rick, personal communication), the heavy atom substructure was determined with SHELXD (33) and the phase problem solved with SHELXE (34). ARPwARP (35), as implemented in CCP4i (36,37), was used to automatically trace the three polypeptide chains in the experimental maps. The structure was anisotropically refined with REFMAC (38) and SHELXL (39), using a thin shells R free set of 893 reflections (2.75% of the total) defined by SHELXPRO (39).…”

Section: Extraction and Purification Of Osteocalcin From Fish Bonymentioning

confidence: 99%

Structural Evidence of a Fourth Gla Residue in Fish Osteocalcin: Biological Implications^,

et al. 2005

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Osteocalcin is a small (45 amino acids) secreted protein found to accumulate in bone and dentin of many organisms by interacting with calcium and hydroxyapatite, through the presence of three γ-carboxylated residues. In this work, we describe the first X-ray crystal structure for a nonmammalian osteocalcin, obtained at 1.4 Å resolution, purified from the marine teleost fish Argyrosomus regius. The three-dimensional fit between the A. regius structure and that of the only other known X-ray structure, the porcine osteocalcin, revealed a superposition of the CR atoms of their metal chelating residues, Gla and Asp, showing that their spatial distribution is consistent with the interatomic distances of calcium cations in the hydroxyapatite crystals. In both structures, the protein forms a tight globular arrangement of their three R-helices while the remaining residues, at N-and C-terminal regions, have essentially no secondary structure characteristics. This study revealed the presence of a fourth γ-carboxylation at Glu 25 , not previously detected in the structure of the porcine osteocalcin or in any other of the sequentially characterized mammalian osteocalcins (human, cow, and rat). A confirmation of the fourth Gla residue in A. regius osteocalcin was achieved via LC-MS analysis. These four doubly charged residues are, together with Asp 24 , concentrated in a common surface region located on the same side of the molecule. This further suggests that the known high affinity of osteocalcin for bone mineral may be derived from the clustering of calcium binding sites on this surface of the molecules.Osteocalcin, also called bone Gla protein or BGP, 1 was the first member of the calcium binding and vitamin K-dependent protein family not associated with blood coagulation to be described, sequenced, and characterized (2, 3). This small protein of 46-50 amino acid residues (depending on the species) (4, 5) is secreted mainly by osteoblasts. It is also one of the most abundant (10-20%) noncollagenous proteins in bones of most vertebrates examined to date, from bony fish to mammals (depending on species, age, and site) (4,(6)(7)(8)(9).Conserved elements in all osteocalcin sequences examined to date include a single disulfide bond (Cys 17 -Cys 23 ) using Argyrosomus regius amino acid sequence numbering, and three γ-carboxyglutamic acid residues (Gla) located at positions 11, 15, and 18 (4, 9-13). These Gla residues are thought to facilitate adsorption of osteocalcin to hydroxyapatite (10,14,15) and are located in the central, conserved portion of the molecule. In contrast, its N-terminal part exhibits considerable sequence variation (4,8,9,13).Initial studies showed that osteocalcin binds strongly to hydroxyapatite crystals (7) and affects mineral formation and mineral crystal growth in solution (16,17). Several studies also suggested a role for osteocalcin as a matrix signal for the recruitment and differentiation of osteoclasts (18-21). However, the understanding of the physiological function of osteocalcin was su...

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“…The structures were refined at the indicated resolution (Table II) using CNS (36) and programs of the CCP4 package (37)(38)(39)(40). Manual adjustments and rebuilding of the models were performed using the program O (41).…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for the Deactivation of the Estrogen-related Receptor γ by Diethylstilbestrol or 4-Hydroxytamoxifen and Determinants of Selectivity

Greschik

Flaig

Renaud

et al. 2004

Journal of Biological Chemistry

120

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The estrogen-related receptor (ERR) ␥ behaves as a constitutive activator of transcription. Although no natural ligand is known, ERR␥ is deactivated by the estrogen receptor (ER) agonist diethylstilbestrol and the selective ER modulator 4-hydroxytamoxifen but does not significantly respond to estradiol or raloxifene. Here we report the crystal structures of the ERR␥ ligand binding domain (LBD) complexed with diethylstilbestrol or 4-hydroxytamoxifen. Antagonist binding to ERR␥ results in a rotation of the side chain of Phe-435 that partially fills the cavity of the apoLBD. The new rotamer of Phe-435 displaces the "activation helix" (helix 12) from the agonist position observed in the absence of ligand. In contrast to the complexes of the ER␣ LBD with 4-hydroxytamoxifen or raloxifene, helix 12 of antagonist-bound ERR␥ does not occupy the coactivator groove but appears to be completely dissociated from the LBD body. Comparison of the ligand-bound LBDs of ERR␥ and ER␣ reveals small but significant differences in the architecture of the ligand binding pockets that result in a slightly shifted binding position of diethylstilbestrol and a small rotation of 4-hydroxytamoxifen in the cavity of ERR␥ relative to ER␣. Our results provide detailed molecular insight into the conformational changes occurring upon binding of synthetic antagonists to the constitutive orphan receptor ERR␥ and reveal structural differences with ERs that explain why ERR␥ does not bind estradiol or raloxifene and will help to design new selective antagonists.The estrogen-related receptors ERR␣, 1 ERR␤, and ERR␥ (NR3B1, -2, and -3) (1) form a subfamily of orphan nuclear receptors that share significant amino acid homology with the estrogen receptors ER␣ and ER␤ (NR3A1 and -2) (2, 3). Because of the high conservation in the DNA binding domain, ERRs and ERs have overlapping DNA binding selectivity (4 -6) and, accordingly, may co-regulate target genes in tissues in which they are co-expressed. ERR subfamily members have for example been shown to modulate the expression of ER target genes in bone (7,8) or breast tissue (9, 10). Importantly, overexpression of ERR␣ and ERR␥ in samples from breast cancer patients correlates with unfavorable and favorable biomarkers, respectively (11). Therefore, these receptors might serve as prognostic markers themselves or even be targets for endocrine therapy in human breast cancer.Despite their significant homology with ERs in the ligand binding domain (LBD), ERRs do not (or only very weakly) respond to estradiol (E2) (2, 12). Furthermore, whereas ERs are ligand-activated receptors, ERRs are constitutively active (13-16), and a structural study confirmed that the ERR␥ LBD can adopt a transcriptionally active conformation and interact with the steroid receptor coactivator 1 (SRC-1) in the absence of any ligand (12). Together, these observations suggest that ERRs are ligand-independent activators of transcription whose activation potential may rather be determined by the presence of transcriptional coactivators (17)(18)(1...

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A graphical user interface to theCCP4 program suite

Cited by 1,208 publications

References 5 publications

Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

Structural Evidence of a Fourth Gla Residue in Fish Osteocalcin: Biological Implications^,

Structural Basis for the Deactivation of the Estrogen-related Receptor γ by Diethylstilbestrol or 4-Hydroxytamoxifen and Determinants of Selectivity

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A graphical user interface to theCCP4 program suite

Cited by 1,208 publications

References 5 publications

Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

Structural Evidence of a Fourth Gla Residue in Fish Osteocalcin: Biological Implications,

Structural Basis for the Deactivation of the Estrogen-related Receptor γ by Diethylstilbestrol or 4-Hydroxytamoxifen and Determinants of Selectivity

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Structural Evidence of a Fourth Gla Residue in Fish Osteocalcin: Biological Implications^,