The Journal of Physical Chemistry B ARTICLE Figure 4. Pair distance distribution, P(r), determined from SAXS data collected on 3 mg/mL MtrA. The maximum dimension, Dmax, is 104 Å, whereas the peak position lies at less than Dmax/2, suggesting that MtrA is elongated. program GNOM,42 I(q) of 3 mg/mL MtrA (over the range q = 0.0174 to 0.2047 Å 1) was converted to a real space function, the pair distance distribution P(r) with a reasonable total GNOM estimate of 0.619 (Figure 4).43 P(r) approaches zero as r approached a value of 104 Å for the maximum chord length, Dmax. The skewed shape of P(r), with a maximum at less than Dmax/2, suggests that the MtrA monomer is an elongated species. Consistent with an elongated shape, the scattering curves plotted as cross-sectional Guinier plots, ln(I(q)*q) versus q2 (Figure 2c), display linear regions. In this representation, the length factor (1/q) is removed, providing information on the cross-sectional size. Linear ﬁts over the range q = 0.069 to 0.086 Å 1, satisfying the q*Rc < 1.3 condition, yielded the cross-sectional radius of gyration, Rc (Figure 2d). Excluded volume and other interpar- ticle eﬀects were removed from Rg and Rc by linear extrapolations of the values determined at 0.5 to 4 mg/mL to zero protein concentration, yielding values of 31.6 ((0.4) and 15.5 ((0.8) Å, respectively. To visualize the overall shape of MtrA, we performed ab initio shape reconstructions on SAXS data collected at 3 mg/mL. Twenty bead models were generated with the simulated anneal- ing program dammif 1.1.0,44 all with reasonable goodness-of-ﬁt to the original data (χ2 = 3.89). A comparison of the original SAXS data and the calculated scattering from a typical model is shown in Figure 5a. The calculated scattering of the model ﬁts the experimental scattering particularly well at q < 0.1 Å 1, where the overall shape information is stored. The apparent discrepancy between the model and experimental data in the high-q region can be attributed to a constant background subtraction imple- mented in dammif to impose a q 4 decay in the Porod region;45 whereas this diﬀerence appears signiﬁcant on a log scale, it is only 0.168 on a linear scale in scattering intensity, or 0.7% of I(0) and is within the errors of background subtraction. Eighteen bead models were chosen by the alignment and averaging program damaver46 with a normalized spatial discrepancy (NSD) of 0.662 (σ2 = 0.047) to produce the ﬁnal averaged model (Figure 5b). The average shape reconstructed by dammif is elongated in one dimension: ∼100 Å in length, consistent with Dmax, and approxi- mately 25 Å 50 Å at the largest cross section. Because MtrA contains both heavy atoms (10 irons) and light atoms (protein and heme rings), we considered whether the usage of uniform-density beads by DAMMIF could aﬀect shape reconstructions, despite minimizing the iron scattering contribu- tions by the use of nonresonant X-ray energy for data collection. Using the program CRYSOL47 and the crystal structure of the pentaheme NrfB, we found that irons contribute more to the overall intensity rather than the shape of the scattering curve, Figure 5. Ab initio shape reconstructions from SAXS data collected on 3 mg/mL MtrA. (a) SAXS data (gray dots) are shown with the calculated scattering of a representative model reconstructed by dammif.44 (b) Molecular surface of the averaged shape reconstructed by dammif44 compared with the NrfB surface smoothed to 15 Å resolution in Chimera.59 particularly at low resolution. Removal of the irons or entire hemes from the NrfB crystal structure leads to 0.2 to 0.3 Å changes in the calculated Rg, with very little change in the shape of the scattering curves up to q = 0.2 Å 1. Such small changes in shape and size are within experimental error and thus are unlikely to aﬀect the shape reconstructions presented here signiﬁcantly. ’ DISCUSSION MtrA is believed to be an essential component in the electron transfer pathway that allows Shewanella oneidensis to interact directly with iron oxides.9,12,15 As one of the few known periplasmic proteins in this electron transfer chain, it likely carries out a critical step in shuttling electrons from the tetraheme cytochrome, CymA, to the outer membrane cytochromes that reduce iron oxide. Here we present the ﬁrst solution-based structural analysis of MtrA using two complementary techniques, SAXS and AUC. Our AUC data indicate that at 0.5 mg/mL MtrA exists as a monomer. The monomeric state of MtrA is supported by our SAXS data, which show that there is no change in size when the concentration of protein is increased to 4 mg/mL. The pair distance distribution P(r) determined by SAXS data suggests that MtrA is elongated in one dimension with a Dmax of 104 Å. It is noted that Dmax is sensitive to the quality of scattering data at low q, where parasitic scattering, interparticle eﬀects, as well as eﬀects of radiation damage dominate.48 Therefore, it is important that Dmax determined in this way be interpreted as an approx- imate quantity. However, the smooth decrease in P(r) to Dmax suggests that the elongated shape of MtrA is due to its inherent shape rather than due to aggregation. We also considered the eﬀect of heme irons on SAXS data analysis using the structure of NrfB as a guide. Calculations suggest that changes to protein scattering curves due to the presence of bound iron atoms are within the experimental error, especially at the X-ray energy used here. Whereas it is outside the scope of this study, it is interesting to consider the use of anomalous iron scattering to characterize heme spatial distributions in multiheme proteins.49 11211 dx.doi.org/10.1021/jp203603r |J. Phys. Chem. B 2011, 115, 11208–11214