Figure 1. Full LigB Ig-like domain region (LigB1-12) determined from experimental SAXS data of 5-domain constructs.

(A) The experimental scattering data for LigB8-12 (black) is shown with the simulated fit (red). The scattering curve is an average of 15 scans ± S.D. (B) The pair distance distributions, P(r), were calculated from SAXS plots of all possible 5-domain LigB Ig-like domains using GNOM. A representation of the 5-domain constructs is shown in the inset. An estimate of construct length based on the longest atomic distances at 20%, 15%, and 10% max population height are shown. (C) DAMFILT envelopes were combined to create a representative envelope of the twelve Ig-like domains. Construct LigB5-9 was not included because the maximum distance distribution exceeds the expected length of a 5-domain construct.

Figure 1—figure supplement 1. SAXS construct design.

(A) Schematic of the full length LigB protein. The set of LigB constructs used to test the optimal number of Ig-like domains and the set of 5-domain constructs used for SAXS analysis. (B) SAXS atomic pair distance distributions, P(r), for 8, 5, 2, and 1 domain constructs (LigB5-12, LigB8-12, LigB11-12, and LigB12, respectively) were calculated from SAXS plots using GNOM. The P(r) was normalized at the first peak (15–20 Å) for each of the distributions. (C) The normalized spatial discrepancy (NSD) derived using the DAMAVER program suite is plotted for multi-domain LigB constructs shown in B. Error bars show S.D. for the for the 10 computed bead models. NSD values > 1 are indicative of heterogeneity in the DAMAVER models.

Figure 1—figure supplement 2. Experimental SAXS data and structures for all LigB Ig-like domain 5-domain constructs.

(A) The experimental scattering data (black line, error bars) are shown for solutions containing LigB1-5, LigB2-6, LigB3-7, LigB4-8, LigB5-9, LigB6-10, LigB7-11, and LigB8-12. The scattering curves are an average of 15 scans ± S.D. A simulated curve (red line) was fit to the data. (B) Guinier plots for the experimental scattering data were calculated with the RAW software using standard cutoffs. Fits (red lines) indicate a short but reasonable linear region characteristic of elongated structures. For data collected on samples after dilution, the relative concentration is indicated as the fraction of original protein stock (3/3, 2/3, 1/3; exact concentrations listed in Figure 1—source data 1). (C) Curves fitted to the SAXS data were used to generate ab initio models for all possible 5-domain stretches of LigB Ig-like domains with DAMMIF. DAMAVER/DAMFILT envelopes, DAMFILT envelopes, and dots depicting domain positions are arranged in rows for the 5-domain constructs. Envelopes were positioned to draw attention to the most significant bends within the stretch of domains. Arrows indicate positions where bending is occurring on the dot representations. (D) The angles created from three neighboring dots within the dot representations were averaged and illustrate the degree to which the neighboring domains deviate from a linear (180°) arrangement.