In-house high-energy-remote SAD phasing using the magic triangle: how to tackle the P1 low symmetry using multiple orientations of the same crystal of human IBA57 to increase the multiplicity

Abstract

This article describes the approach used to solve the structure of human IBA57 in-house by 5-amino-2,4,6-triiodoisophthalic acid (I3C) high-energy-remote single-wavelength anomalous dispersion (SAD) phasing. Multiple orientations of the same triclinic crystal were exploited to acquire sufficient real data multiplicity for phasing. How the collection of an in-house native data set and its joint use with the I3C derivative through a SIRAS approach decreases the data multiplicity needed by almost 50% is described. Furthermore, it is illustrated that there is a clear data-multiplicity threshold value for success and failure in phasing, and how adding further data does not significantly affect substructure solution and model building. To our knowledge, this is the only structure present in the PDB that has been solved in-house by remote SAD phasing in space group P1 using only one crystal. All of the raw data used, derived from the different orientations, have been uploaded to Zenodo in order to enable software developers to improve methods for data processing and structure solution, and for educational purposes.

Structural properties of [2Fe-2S] ISCA2-IBA57: a complex of the mitochondrial iron-sulfur cluster assembly machinery

“…SAXS data on IBA57 and apo ISCA2 showed that the two proteins are, respectively, monomeric and dimeric in solution, in agreement with previous studies 18,21 . The SAXS data are in agreement with the solved crystal structure of IBA57 21,33 , indicating that this structural arrangement is preserved in solution. Since no structure is available for dimeric apo ISCA2, structural models were obtained either by SAXS-guided rigid-body modeling of the dimer of ISCA2 from monomeric ISCA2 structural model or by imposing the same dimerization as in the crystal structures of bacterial ISCA homologues.…”

“…The SAXS data on IBA57 suggested that the crystal structure (6QE3.pdb) 21,33 is maintained in solution and the scattering computed from this structure fits well the experimental SAXS profile of IBA57 (discrepancy χ 2 = 1.075, continuous line in Fig. 1a).…”

“…While the structure of IBA57 is available 21,33 , the structure of human [2Fe-2S]-bound ISCA2 is not. However, a [2Fe-2S] cluster-bound crystal structure of IscA from Thermosynechococcus elongatus has been solved 35 .…”

“…Structural model of human IBA57 was already available in the Protein Data Bank (PDB ID 6QE3) 21,33 . An experimentally validated structure of monomeric apo ISCA2 was already available 18 .…”

Molecular Basis of Rare Diseases Associated to the Maturation of Mitochondrial [4Fe-4S]-Containing Proteins

“…Considering the large number of pathogenic missense mutations found in IBA57, i.e., twenty-four mutations ( Table 1 ), we have grouped them on the basis of their solvent accessibility and analyzed their impact on the structures of IBA57 [ 28 , 107 ] and of the [2Fe-2S] IBA57-ISCA2 complex [ 29 ]. The solvent accessibility was obtained with the program NACCESS applied to the available crystallographic structure of IBA57, considering a residue as solvent-exposed when the relative solvent accessibility of the side-chain is over 40%.…”