Single-site mutants in the Cu,Zn superoxide dismutase (SOD) gene ( SOD1 ) occur in patients with the fatal neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). Complete screening of the SOD1 coding region revealed that the mutation Ala ⁴ to Val in exon 1 was the most frequent one; mutations were identified in exons 2, 4, and 5 but not in the active site region formed by exon 3. The 2.4 Å crystal structure of human SOD, along with two other SOD structures, established that all 12 observed FALS mutant sites alter conserved interactions critical to the β-barrel fold and dimer contact, rather than catalysis. Red cells from heterozygotes had less than 50 percent normal SOD activity, consistent with a structurally defective SOD dimer. Thus, defective SOD is linked to motor neuron death and carries implications for understanding and possible treatment of FALS.

Crystallography supplies unparalleled detail on structural information critical for mechanistic analyses; however, it is restricted to describing low energy conformations of macromolecules within crystal lattices. Small angle X-ray scattering (SAXS) offers complementary information about macromolecular folding, unfolding, aggregation, extended conformations, flexibly linked domains, shape, conformation, and assembly state in solution, albeit at the lower resolution range of about 50 Å to 10 Å resolution, but without the size limitations inherent in NMR and electron microscopy studies. Together these techniques can allow multi-scale modeling to create complete and accurate images of macromolecules for modeling allosteric mechanisms, supramolecular complexes, and dynamic molecular machines acting in diverse processes ranging from eukaryotic DNA replication, recombination and repair to microbial membrane secretion and assembly systems. This review addresses both theoretical and practical concepts, concerns and considerations for using these techniques in conjunction with computational methods to productively combine solution scattering data with high-resolution structures. Detailed aspects of SAXS experimental results are considered with a focus on data interpretation tools suitable to model protein and nucleic acid macromolecular structures, including membrane protein, RNA, DNA, and protein–nucleic acid complexes. The methods discussed provide the basis to examine molecular interactions in solution and to study macromolecular flexibility and conformational changes that have become increasingly relevant for accurate understanding, simulation, and prediction of mechanisms in structural cell biology and nanotechnology.

Modern small-angle scattering (SAS) experiments with X-rays or neutrons provide a comprehensive, resolution-limited observation of the thermodynamic state. However, methods for evaluating mass and validating SAS-based models and resolution have been inadequate. Here we define the volume of correlation, V_c, a SAS invariant derived from the scattered intensities that is specific to the structural state of the particle, but independent of concentration and the requirements of a compact, folded particle. We show that V_c defines a ratio, Q_R, that determines the molecular mass of proteins or RNA ranging from 10 to 1,000 kilodaltons. Furthermore, we propose a statistically robust method for assessing model-data agreements (χ²_free) akin to cross-validation. Our approach prevents over-fitting of the SAS data and can be used with a newly defined metric, R_SAS, for quantitative evaluation of resolution. Together, these metrics (V_c, Q_R, χ²_free and R_SAS) provide analytical tools for unbiased and accurate macromolecular structural characterizations in solution.