<i>SHARP</i>: maximum-likelihood refinement of heavy-atom parameters in the MIR and MAD methods

Fortelle, Eric de La; Bricogne, G.

doi:10.1107/s0108767396096808

Cited by 108 publications

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“…The data set was indexed, integrated and scaled with the XDS package 55 . Heavy atom location and phasing were performed with the program SHARP 56 . Furthermore, the program SOLOMON 57 was used for phase improvement by solvent flipping.…”

Section: Methodsmentioning

confidence: 99%

An artificial PPR scaffold for programmable RNA recognition

et al. 2014

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Pentatricopeptide repeat (PPR) proteins control diverse aspects of RNA metabolism in eukaryotic cells. Although recent computational and structural studies have provided insights into RNA recognition by PPR proteins, their highly insoluble nature and inconsistencies between predicted and observed modes of RNA binding have restricted our understanding of their biological functions and their use as tools. Here we use a consensus design strategy to create artificial PPR domains that are structurally robust and can be programmed for sequence-specific RNA binding. The atomic structures of these artificial PPR domains elucidate the structural basis for their stability and modelling of RNA-protein interactions provides mechanistic insights into the importance of RNA-binding residues and suggests modes of PPR-RNA association. The modular mode of RNA binding by PPR proteins holds great promise for the engineering of new tools to target RNA and to understand the mechanisms of gene regulation by natural PPR proteins.

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Section: Methodsmentioning

confidence: 99%

An artificial PPR scaffold for programmable RNA recognition

et al. 2014

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“…12 Use of SHARP 13 for phase refinement and density modification yielded an experimental electron density map suitable for use with ARP/wARP 14 for automated model building. 93% of the polypeptide chain was built without manual intervention.…”

Section: Crystallization Data Collection and Structure Determinationmentioning

confidence: 99%

X‐ray crystal structure of the B component of Hemolysin BL from Bacillus cereus

Madegowda

Eswaramoorthy

Burley³

et al. 2008

Proteins

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“…The missing two sites correspond to a disordered loop in both monomers. Sites were refined using program MLPHARE (Otwinowski 1991) and later using program SHARP (La Fortelle et al 1997). Table 2 gives phasing statistics for the oxidized data set.…”

Section: Phasing and Structure Solutionmentioning

confidence: 99%

Crystal structure of threonine synthase from Arabidopsis thaliana

Thomazeau

2001

Protein Science

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Threonine synthase (TS) is a PLP-dependent enzyme that catalyzes the last reaction in the synthesis of threonine from aspartate. In plants, the methionine pathway shares the same substrate, O-phospho-Lhomoserine (OPH), and TS is activated by S-adenosyl-methionine (SAM), a downstream product of methionine synthesis. This positive allosteric effect triggered by the product of another pathway is specific to plants. The crystal structure of Arabidopsis thaliana apo threonine synthase was solved at 2.25 Å resolution from triclinic crystals using MAD data from the selenomethionated protein. The structure reveals a fourdomain dimer with a two-stranded ␤-sheet arm protruding from one monomer onto the other. This domain swap could form a lever through which the allosteric effect is transmitted. The N-terminal domain (domain 1) has a unique fold and is partially disordered, whereas the structural core (domains 2 and 3) shares the functional domain of PLP enzymes of the same family. It also has similarities with SAM-dependent methyltransferases. Structure comparisons allowed us to propose potential sites for pyridoxal-phosphate and SAM binding on TS; they are close to regions that are disordered in the absence of these molecules.Keywords: Allostery; MAD; pyridoxal phosphate; S-adenosyl-methionine; threonine synthesis Aspartate is a precursor for the synthesis of several other amino acids, including lysine, threonine, isoleucine, and methionine. Threonine synthase (TS) (EC 4.2.99.2) is the last enzyme of the threonine synthesis pathway (Fig. 1A). It is a PLP-dependent enzyme and a 110-kD homodimer in solution. It catalyzes the conversion of O-phospho-L-homoserine (OPH) into threonine and inorganic phosphate (Fig. 1B). The bacterial reaction was studied by following the absorbence of PLP and was shown to present at least seven steps (Laber et al. 1994).In plants, OPH is a branch point intermediate between the methionine/S-adenosyl-methionine (SAM) and threonine/ isoleucine pathways (Giovanelli et al. 1984). Thus TS competes for OPH with cystathionine-␥-synthase (CGS), the first committed enzyme in the methionine pathway (Fig. 1A). Flux regulation between the threonine and methionine pathways does not result from end-product inhibition but, rather, via a strong stimulation of TS by SAM (Giovanelli et al. 1984). Indeed, previous results showed that the allosteric binding of SAM leads to an eightfold increase in the rate of catalysis and to a 25-fold decrease in the K M value for OPH (Curien et al. 1998). Saturation isotherms of TS by SAM disclosed positive cooperativity and indicated that at least two SAM molecules can bind on each dimeric enzyme (Curien et al. 1998). From these results, it was concluded that the dramatic modification in kinetic properties of plant TS originates from an allosteric and cooperative transition that is induced by SAM. Furthermore, this transition occurs at a

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SHARP: maximum-likelihood refinement of heavy-atom parameters in the MIR and MAD methods

Cited by 108 publications

References 0 publications

An artificial PPR scaffold for programmable RNA recognition

An artificial PPR scaffold for programmable RNA recognition

X‐ray crystal structure of the B component of Hemolysin BL from Bacillus cereus

Crystal structure of threonine synthase from Arabidopsis thaliana

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