Propagating errors in small-angle scattering data treatment

Svergun, D. I.; Pedersen, Jan Skov

doi:10.1107/s0021889893008337

Cited by 44 publications

(46 citation statements)

References 7 publications

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“…Calculation of the distance distribution functions p(r) and desmearing of the intensity were performed with the method of indirect Fourier transformation using the program GNOM, which also delivers errors for the p(r) curves (22,23).…”

Section: Methodsmentioning

confidence: 99%

The Allosteric Effector l-Lactate Induces a Conformational Change of 2×6-meric Lobster Hemocyanin in the Oxy State as Revealed by Small Angle X-ray Scattering

Hartmann

Lohkamp

Hellmann

et al. 2001

Journal of Biological Chemistry

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Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector L-lactate on the oxygenated quaternary structure of the 2؋6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mM L-lactate resulted in different scattering curves compared with those obtained in the absence of L-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of L-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves.Hemocyanins are the oxygen transport proteins of most molluscs and arthropods. They are enormous molecular structures, ranging in molecular mass from 4.5 ϫ 10 5 to more than 10 7 Da; some contain more than 100 oxygen binding sites (1-3). Hemocyanins exhibit cooperative oxygen binding and respond to a variety of allosteric effectors (4 -8). As a consequence of their structural complexity, hemocyanins have proved important in extending our understanding of allostery. For example, explanation of the cooperative oxygen binding by arthropod hemocyanin has required extension of the classical MWC 1 model to the "nesting" model (8, 9), which reflects the hierarchical structure of these proteins. The nesting model requires four states (rT, rT, tR, rR) instead of the two states (T, R) of the MWC model. It makes the prediction that even fully oxygenated hemocyanin can exist in two conformational states. Although the model has served well to explain oxygen binding and its dependence on effector (see Ref. 8, for example), it has never been tested directly. That is, no experiments have been performed to detect the two oxy conformations predicted by the nesting model. To be definitive, such experiments must be carried out in solution, under rigorously controlled oxygenation levels and with a sensitivity sufficient to detect even small conformational changes.A suitable method to detect conformational changes of large proteins in solution is small angle x-ray scattering (SAXS). This technique has been applied successfully to monitor changes of the quaternary structure for several cooperative proteins such as hemoglobin and a number of allosteric enzymes (10 -18). Different conformations have also been reported for the oxy-and deoxyhemocyanin from the tarantula Eurypelma californicum fro...

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Section: Methodsmentioning

confidence: 99%

The Allosteric Effector l-Lactate Induces a Conformational Change of 2×6-meric Lobster Hemocyanin in the Oxy State as Revealed by Small Angle X-ray Scattering

Hartmann

Lohkamp

Hellmann

et al. 2001

Journal of Biological Chemistry

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“…The errors on p(r) and the parameters d(q ϭ 0)/d⍀ and R g,av that were derived from p(r) were estimated by the Monte Carlo procedure as described by Svergun and Pedersen. 44 Because of particle interactions, some of the p(r) functions determined for the ␤-lg samples give negative values for large values of r. This effect on p(r) is due to the structure factor [Eq. (4)] that influences the scattering data at low q values.…”

Section: Indirect Fourier Transformation Of Sans Datamentioning

confidence: 99%

Association behavior of native ?-lactoglobulin

Verheul¹,

Pedersen

Roefs³

et al. 1999

Biopolymers

196

173

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“…Hansen & Wilkins, 1994). Systematic errors like these and those mentioned previously from the unknown amount of aggregation will dominate the error on the estimates (see also Svergun & Pedersen, 1994).…”

Section: Maximum Entropymentioning

confidence: 70%

A Small-Angle X-ray Scattering Study of the Binding of Cyclosporin A to Cyclophilin

Knott¹,

Capel²,

Hansen³

et al. 1995

J Appl Cryst

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The small-angle X-ray scattering (SAXS) technique was used to investigate structural characteristics of the protein cyclophilin in solution and to attempt to detect major changes induced by the binding of the immunosuppressant drug cyclosporin A. Maximum-entropy methods were used to analyse the experimental SAXS data. The measured radius of gyration, Rg, for cyclophilin is 16.3 (5) A. This is equivalent to a compact sphere of radius 21.0/~. There is qualitative agreement between the experimental SAXS profiles and the derived distancedistribution function, p(r), for cyclophilin, and similar profiles calculated from the crystallographic structure. The notable discrepancy is the difference of approximately 1.5 A in the estimated radius of the equivalent sphere. On binding cyclosporin A, the main structurerelated change in cyclophilin observed under these experimental conditions is an increased propensity to form oligomers. Meaningful estimates of Rg for the monomeric complex are not possible because of the presence of a significant population of aggregates. In a second series of experiments, both native cyclophilin and the cyclophilin/cyclosporin A complex readily formed aggregates under the prevailing experimental conditions.

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Propagating errors in small-angle scattering data treatment

Cited by 44 publications

References 7 publications

The Allosteric Effector l-Lactate Induces a Conformational Change of 2×6-meric Lobster Hemocyanin in the Oxy State as Revealed by Small Angle X-ray Scattering

The Allosteric Effector l-Lactate Induces a Conformational Change of 2×6-meric Lobster Hemocyanin in the Oxy State as Revealed by Small Angle X-ray Scattering

Association behavior of native ?-lactoglobulin

A Small-Angle X-ray Scattering Study of the Binding of Cyclosporin A to Cyclophilin

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