Locating the anomalous scatterer substructures in halide and sulfur phasing

Abstract

Improved data quality now makes it feasible to exploit the weak anomalous signal derived only from the sulfurs inherent to the protein or in particular from halide ions incorporated by soaking. The latter technique requires the location of a high number of partially occupied halide sites. This number appears to be roughly proportional to the exposed protein surface. This paper explores the application of dual-space ab initio methods as implemented in the program SHELXD to the location of substructures of sulfur in SAD experiments, bromide in SAD and MAD experiments and iodide using SAD and SIRAS to determine the anomalous-atom substructure. Sets of atoms consistent with the Patterson function were generated as a starting point for the dual-space recycling procedure in SHELXD. The substructure is then expanded to the full structure by maximum-likelihood phasing with SHARP and density modification with the program DM. Success in the location of the substructures and subsequent phasing depends critically on the quality of the data and on the extent of the anomalous signal. This varies with each crystal and soak, but for the same crystal the significance of the anomalous signal was found to be highly sensitive to the redundancy of the intensity measurements, which in some cases made all the difference. This is illustrated by the determination of the previously unknown structure of repeat 11 of the human mannose-6-phosphate/insulin-like growth factor II receptor (Man6P/IGFII-receptor), with 310 amino acids in the asymmetric unit, which was phased by soaking the crystals in a cryoprotectant solution containing halide anions.

Structural Insights into Endosomal Sorting Complex Required for Transport (ESCRT-I) Recognition of Ubiquitinated Proteins

“…However, using a highly redundant, lower resolution single anomalous dispersion (SAD) data set collected at SRS 14.1, eight Se sites were readily located using the program SnB (44 -46) and subsequently refined with autoSHARP (47). The greater efficiency of determining substructures with high multiplicity SAD data is a common observation (48,49 using difference Fourier maps. The 2.3 Å resolution MAD phases were then refined by SHARP (Table I).…”

Kinetics of Insulin-like Growth Factor II (IGF-II) Interaction with Domain 11 of the Human IGF-II/Mannose 6-phosphate Receptor: Function of CD and AB Loop Solvent-exposed Residues

“…[51][52][53][54][55] The crystal structure of IGF2R domain 11 has been solved at 1.4 Å resolution using anomalous scattering of sulphur, 7 and has been confirmed by others. 56 The domain 11 IGF2R structure reveals two hydrophobic sites on the surface of domain 11, the first that identifies the putative IGF-II-binding site within the cleft of the b-barrel structure, spatially analogous to the hydrophilic sugar-binding site of the CD-MPR, and a second that is implicated in domain-domain interactions. 7,57 The IGF-II-binding pocket (400 Å 3 ) is formed by the b-strands A, B, C and D and the loops AB, CD and FG, with shorter loops conferring a shallower binding cavity than that of the CD-MPR.…”

Cation-independent Mannose 6-Phosphate Receptor

“…In addition, the crystal structure of seven (i.e. domains 1-3, domains [11][12][13][14] of the 15 domains of the CI-MPR has been determined to date, which shows that each of these MRH domains shares a similar fold (21)(22)(23). Its ability to function as an efficient cargo transporter is likely due to the fact that the CI-MPR contains three Man-6-P binding sites: essential residues for carbohydrate recognition are located within domains 3, 5, and 9 (24 -30) (see Fig.…”