Computational protocol: A general method for the unbiased improvement of solution NMR structures by the use of related X-Ray data, the AUREMOL-ISIC algorithm

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Protocol publication

[…] The calculation of a dense network of dihedral angle and distance restraints with the PERMOL-algorithm from bundles of structures has been described earlier [,]. and is implemented in AUREMOL []. Here, the expectation values and standard deviations are calculated. Error ranges are approximated from the standard deviations on the basis of the t-test. In case the original set contains only one structure the corresponding structural bundle has to be calculated first. In this regard we will discuss in the following only the most important case of crystal structures that are usually represented as distinct single structures Sip (p = 1). But the principle can be applied to other data.Depending on the unit cell and the refinement method used sometimes more than one structure is deposited in the data base (p > 1). However, even then the statistical ensemble is too small. The solution to this problem is that in analogy to the calculation of NMR-structures the inherent coordinate uncertainties can be used to calculate structural bundles and from those a set of substitute restraints Ri* is obtained. Therefore, we first determine a set of restraints Rix* that represent the original X-ray structure(s) from inter-atomic distances and dihedral angles in the crystal structure(s) together with the corresponding coordinate uncertainties. Using these restraints a set of structures Six is created, from which the set of substitute restraints Ri* is created using PERMOL. For generating the set Rix* two factors that are usually published together with the structure that can be used for a conservative estimate of the structural variations. In a first approximation the expected average error in atomic positions σ(r0) is about 1/3 of the resolution R []. In a more involved analysis σ(rm) of the atoms m possessing low B-factors is often estimated from Luzzati plots. Second the local B-factors can be used to introduce additional errors for specific atoms possessing significant B-values. Static and thermal disorder can effectively spread out the electron density of a given atom mand this increases its B-factor. The B-factor is related to the rms error in the position of an atom by the equation: σ ( r m ) = B m 8 ⋅ π 2       ( 4 ) [email protected]@[email protected]@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaaiiGacqWFdpWCcqGGOaakieqacqGFYbGCdaWgaaWcbaGaemyBa0gabeaakiabcMcaPiabg2da9maakaaabaWaaSaaaeaacqWGcbGqdaWgaaWcbaGaemyBa0gabeaaaOqaaiabiIda4iabgwSixlab=b8aWnaaCaaaleqabaGaeGOma[email protected][email protected] Bm denotes the B-factor of a given atom m and σ(rm) is the corresponding average error in atom positions.Since for the calculations a conservative estimate of distances ranges is most useful, the square of the standard deviation σ2(dm,n) of the distance dm,n between two atoms m and n (m | n) is approximated byσ2(dm,n) = σ(rm)2 + σ(rn)2 + 2σ(r0)2     (5)For a more detailed description on the precision of protein structures see the article by Cruickshank []. When more than one structure of the same crystal is contained in the data base they can be considered as separate structural sets Si and handled in an analogous way. As mentioned above, using this preliminary set of restraints Rix* a bundle of structures Six is calculated by employing programs such as DYANA [], XPLOR-NIH [] or CNS []. From this bundle a set of restraints Ri* is calculated in the same way as it has been done for the restraint set R1 of the leading structure S1. [...] NMR data evaluation was performed with the program AUREMOL (V 2.2.1). Expectation values and standard deviations of cyclic quantities were calculated according to Döker et al., []. Sequence alignment was performed with a module for pair-wise sequence alignment based on the Needleman-Wünsch algorithm and the BLOSUM62 matrix that we recently included in the AUREMOL module PERMOL [,]. The resulting refined solution structures were validated on the experimental NMR data by the calculation of NMR R-factors []. For investigating the stereo-chemical quality PROCHECK-NMR was employed [] and rmsd values were calculated using MOLMOL []. […]

Pipeline specifications

Software tools Xplor-NIH, CNS, PROCHECK, MOLMOL
Applications NMR-based proteomics analysis, Protein structure analysis
Organisms Dipturus trachyderma, Schizosaccharomyces pombe, Homo sapiens