Developing advanced X-ray scattering methods combined with crystallography and computation

Abstract

The extensive use of small angle x-ray scattering (SAXS) over the last few years is rapidly providing new insights into protein interactions, complex formation and conformational states in solution. This SAXS methodology allows for detailed biophysical quantification of samples of interest. Initial analyses provide a judgment of sample quality, revealing the potential presence of aggregation, the overall extent of folding or disorder, the radius of gyration, maximum particle dimensions and oligomerization state. Structural characterizations include ab initio approaches from SAXS data alone, and when combined with previously determined crystal/NMR, atomistic modeling can further enhance structural solutions and assess validity. This combination can provide definitions of architectures, spatial organizations of protein domains within a complex, including those not determined by crystallography or NMR, as well as defining key conformational states of a protein interaction. SAXS is not generally constrained by macromolecule size, and the rapid collection of data in a 96-well plate format provides methods to screen sample conditions. This includes screening for co-factors, substrates, differing protein or nucleotide partners or small molecule inhibitors, to more fully characterize the variations within assembly states and key conformational changes. Such analyses may be useful for screening constructs and conditions to determine those most likely to promote crystal growth of a complex under study. Moreover, these high throughput structural determinations can be leveraged to define how polymorphisms affect assembly formations and activities. This is in addition to potentially providing architectural characterizations of complexes and interactions for systems biology-based research, and distinctions in assemblies and interactions in comparative genomics. Thus, SAXS combined with crystallography/NMR and computation provides a unique set of tools that should be considered as being part of one’s repertoire of biophysical analyses, when conducting characterizations of protein and other macromolecular interactions.

RNF 4 interacts with both SUMO and nucleosomes to promote the DNA damage response

“…These predicted interactions are centered at the RKK basic cluster, in addition to a neighboring set of basic residues, His 182 -Lys 183 -Arg 184 , occurring in hRNF4 but not RING1b and RNF168 ( Fig 1C and Supplementary Fig S2A). Using in-solution SAXS [32], we determined that hRNF4 RING domain forms a dimer in solution. Interestingly, the RNF4 structures with the C-terminus in a folded conformation, either apo-protein (PDB code 2XEU) or as observed in a complex with UbcH5:ubiquitin (4AP4), are a closer fit to the in-solution SAXS experimental data than RNF4 structures with the C-terminus in the extended conformation ( Supplementary Fig S2).…”

Techniques for the analysis of pentacyclic triterpenoids in medicinal plants

“…X‐ray spectroscopy is an advanced technique used for the analysis of absolute configuration of pentacyclic triterpenoids in medicinal plants. Given that the densities of materials vary, the internal structure of compounds can be measured by recording the changes in the light intensity of X‐ray . Froelich et al.…”

“…X-ray spectroscopy is an advanced technique used for the analysis of absolute configuration of pentacyclic triterpenoids in medicinal plants. Given that the densities of materials vary, the internal structure of compounds can be measured by recording the changes in the light intensity of X-ray [128]. Froelich et al [129] used this method to determine the accurate molecular structure of 3β-acetoxy-12-hydroxyimino-18b-oleanan-28,13b-olide, 3β-acetoxy-12-nitrile-12,13-seco-15(14→13)-abeoolean-14(27)-en-28,13b-olide, and 3β-acetoxy-12-oxo-12a-aza-C-homoolean-13(18)-en-28-oic acid.…”

Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

“…The inferred oligomeric state of holoNarGH and variants in solution, confirmed by determination of the volume of the protein by DAMMIF, was found to be different from the X-ray crystal structure. SAXS can indeed reveal different solution behavior compared to the crystalline assembly, probably because of crystal packing or altered intermolecular interactions due to the high solute concentrations in the crystallization solution60. Ab initio shape determination was performed using DAMMIF61.…”