1,721,033 research outputs found
The signal-to-noise ratio in SAD experiments
No full textDe novo single-wavelength anomalous dispersion (SAD) determination of macromolecular structures requires accurate measurement of anomalous signal from naturally occurring (S, P, Ca, etc.) or incor- porated (Se, Hg, etc.) anomalous scatterers. The quality and the level of the anomalous signal in SAD datasets can be assessed using, as an indicator, the average anomalous signal-to-noise level ⟨|Ianom|/σ (I)⟩ based on intensities or ⟨|Fanom|/σ (F)⟩ based on amplitudes. The value for the average anomalous signal-to-noise for a successful SAD experiment is a matter of debate given its high vari- ability from one SAD experiment to another. We present a mathemat- ical model relating the ⟨|Ianom|/σ(I)⟩ to the overall final ⟨I/σ(I)⟩, necessary for successful SAD phasing. By statistical analysis of the data from 115 successful native SAD experiments, as available in the PDB, we show that the experimental values of ⟨|Ianom|/σ(I)⟩ fol- low a Gamma distribution with an average value of 1.2±0.3. This mathematical model allows a-priori prediction of the overall ⟨I/σ (I)⟩, necessary for successful SAD phasing given the description of the sample expressed. The results reported here are of general appli- cability to any phasing experiment involving the measurement of anomalous signal from any anomalous scatterer or X-ray source, including X-FEL data
The interdependence of wavelength, redundancy and dose in sulfur SAD experiments
No full textIn the last decade, the popularity of sulfur SAD anomalous dispersion experiments has spread rapidly among synchrotron users as a quick and streamlined way of solving the phase problem in macromolecular crystallography. On beamline 10 at SRS (Daresbury Laboratory, UK), a versatile design has allowed test data sets to be collected at six wavelengths between 0.979 and 2.290 Å in order to evaluate the importance and the interdependence of experimental variables such as the Bijvoet ratio, wavelength, resolution limit, data redundancy and absorbed X-ray dose in the sample per data set. All the samples used in the experiments were high-quality hen egg-white lysozyme crystals. X-radiation damage was found to affect disulfide bridges after the crystals had been given a total dose of 0.20 Å 107 Gy. However, with such a total dose, it was still possible in all cases to find a strategy to collect data sets to determine the sulfur substructure and produce good-quality phases by choosing an optimum combination of wavelength, exposure time and redundancy. A 〈|Δano|/δ(Δ ano)〉 greater than 1.5 for all resolution shells was a necessary requirement for successful sulfur SAD substructure location. Provided this is achieved, it seems possible to find an optimum compromise between wavelength, redundancy and dose to provide phasing information. The choice of the wavelength should then follow the sample composition and the diffracting properties of the crystal. For strongly diffracting crystals, wavelengths equal or shorter than 1.540 Å can be selected to capture the available data (provided the Bijvoet ratio is reasonable), while a longer wavelength, to gain as high a Bijvoet ratio as possible, must be used for more weakly diffracting crystals. These results suggest that an approach to a sulfur SAD experiment based on a complete description of the crystal system and the instrument for data collection is useful. © International Union of Crystallography 2008
Expression, purification, crystallization and preliminary X-ray analysis of glucose-1-phosphate uridylyltransferase (GalU) from Erwinia amylovora
No full textCloning, expression, purification, crystallization and preliminary X-ray analysis of Ea Lsc, a levansucrase from Erwinia amylovora
No full textOpen and closed states of Candida antarctica lipase B: protonation and the mechanism of interfacial activation
No full textLipases (EC 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates, such as triacylglycerols, phospholipids, and other insoluble substrates, acting in aqueous as well as in low-water media, thus being of considerable physiological significance with high interest also for their industrial applications. The hydrolysis reaction follows a two-step mechanism, or “interfacial activation,” with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Among lipases, Candida antarctica lipase B (CALB) has never shown any significant interfacial activation, and a closed conformation of CALB has never been reported, leading to the conclusion that its behavior was due to the absence of a lid regulating the access to the active site. The lid open and closed conformations and their protonation states are observed in the crystal structure of CALB at 0.91 Å resolution. Having the open and closed states at atomic resolution allows relating protonation to the conformation, indicating the role of Asp145 and Lys290 in the conformation alteration. The findings explain the lack of interfacial activation of CALB and offer new elements to elucidate this mechanism, with the consequent implications for the catalytic properties and classification of lipases
Extensive counter-ion interactions seen at the surface of subtilisin in an aqueous medium
No full textThe extent of protein and counter-ion interactions in solution is still far from being fully described and understood. In low dielectric media there is documented evidence that counter-ions do bind and affect enzymatic activity. However, published crystal structures of macromolecules of biological interest in aqueous solution often do not report the presence of any counter-ions on the surface. The extent of counter-ion interactions within subtilisin in an aqueous medium has been investigated crystallographically using CsCl soak and X-ray wavelength optimised anomalous diffraction at the Cs K-edge. Ten Cs+, as well as six Cl- sites, have been clearly identified, revealing that in aqueous salt solutions ions can bind at defined points around the protein surface. The counter-ions do not generally interact with formal charges on the protein; formally neutral oxygens, mostly backbone carbonyls, mostly coordinate the Cs+ ions. The Cl- ion sites are also found likely to be near positive charges on the protein surface. The presence of counter-ions substantially changes the protein surface electrical charge. The surface charge distribution on a protein is commonly discussed in relation to enzyme function. The correct identification of counter-ions associated with a protein surface is necessary for a proper understanding of an enzyme's function
Exploring the conformational space of the mobile flap in Sporosarcina pasteurii urease by cryo-electron microscopy
Full text linkTo fully understand enzymatic dynamics, it is essential to explore the complete conformational space of a biological catalyst. The catalytic mechanism of the nickel-dependent urease, the most efficient enzyme known, holds significant relevance for medical, pharmaceutical, and agro-environmental applications. A critical aspect of urease function is the conformational change of a helix-turn-helix motif that covers the active site cavity, known as the mobile flap. This motif has been observed in either an open or a closed conformation through X-ray crystallography studies and has been proposed to stabilize the coordination of a urea molecule to the essential dinuclear Ni(II) cluster in the active site, a requisite for subsequent substrate hydrolysis. This study employs cryo-electron microscopy (cryo-EM) to investigate the transient states within the conformational space of the mobile flap, devoid of the possible constraints of crystallization conditions and solid-state effects. By comparing two cryo-EM structures of Sporosarcina pasteurii urease, one in its native form and the other inhibited by N-(n-butyl) phosphoric triamide (NBPTO), we have unprecedently identified an intermediate state between the open and the catalytically efficient closed conformation of the helix-turn-helix motif, suggesting a role of its tip region in this transition between the two states
Inhibition of Urease by Hydroquinones: A Structural and Kinetic Study
Full text linkHydroquinones are a class of organic compounds abundant in nature that result from the full reduction of the corresponding quinones. Quinones are known to efficiently inhibit urease, a Ni(II)‐containing enzyme that catalyzes the hydrolysis of urea to yield ammonia and carbonate and acts as a virulence factor of several human pathogens, in addition to decreasing the efficiency of soil organic nitrogen fertilization. Here, we report the molecular characterization of the inhibition of urease from Sporosarcina pasteurii (SPU) and Canavalia ensiformis (jack bean, JBU) by 1,4‐hydroquinone (HQ) and its methyl and tert‐butyl derivatives. The 1.63‐Å resolution X‐ray crystal structure of the SPU‐HQ complex discloses that HQ covalently binds to the thiol group of αCys322, a key residue located on a mobile protein flap directly involved in the catalytic mechanism. Inhibition kinetic data obtained for the three compounds on JBU reveals the occurrence of an irreversible inactivation process that involves a radical‐based autocatalytic mechanism
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