C O M M U N I C A T I O N S Figure 2. Comparison of the 15N-13CR region of correlation spectra obtained with ZF-TEDOR mixing for two differently labeled 2m ﬁbril samples. (a) 2- 2m, τmix ) 1.6 ms, 12 mg of sample, 2 days of experimental time. Labels correspond to intramolecular Ni-CRi transfer, unless otherwise noted, while labels in gray denote cross-peaks that appear only in the 2- 2m spectrum. (b) Mixed 2- 2m, τmix ) 16 ms, 16 mg of sample, 9 days of experimental time. Labels correspond to intermolecular Ni-CRi or Ni-CRi-1 transfer, unless otherwise noted. distances of ∼5.0-5.5 Å. The maximum bulk transfer efﬁciency for the CR region is ∼3%, which is better than the experimental transfer efﬁciencies observed for uniformly 13C labeled samples (<1% for similar distances).20 In order to obtain site-speciﬁc information regarding the origin of the 15N-13C intermolecular contacts in mixed 2- 2m, we recorded a 2D ZF-TEDOR experiment with τmix ) 16 ms (Figure S3). This spectrum presents excellent resolution (13C line widths ∼50 Hz) and sufﬁcient sensitivity after a long acquisition period, which was facilitated by the robustness of the TEDOR sequence. Overall, the positions of the observed cross-peaks in this mixed 2- 2m spectrum correspond exactly with the positions of cross-peaks in one-bond (Figure 2a) or two-bond TEDOR spectra (data not shown) of a 2m ﬁbril sample prepared from 100% 15N, 2-13C glycerol labeled material (2- 2m). The majority of the cross-peaks in the mixed 2- 2m sample could be readily assigned based on known chemical shifts of long, straight 2m ﬁbrils,10 and they correspond exclusively to intermolecular Ni-CRi, Ni-CRi-1, Ni-COi, or Ni-COi-1 transfer (Figures 2b and S3). In particular, the following residues giving rise to intermolecular contacts in the mixed 2- 2m sample were assigned: H31-S33, N42, G43, R45, I46, V49, H51-F62, P72, T73, and Y78-V82. While P32, S33, G43, F56, S57, K58, and F62 are part of well-ordered loops in the ﬁbrils, the remainder of the residues represent all of the currently assigned ﬁbril -strands. Some cross-peaks in Figure 2b (mixed 2- 2m) do not presently have assignments. Conversely, not all of the strong cross-peaks shown in Figure 2a (2- 2m) appear in the mixed 2- 2m spectrum. This includes G18, G29, E44, H84, and V85 (shown in gray in Figure 2a) among others. This is most likely due to differences in 17078 J. AM. CHEM. SOC. 9 VOL. 132, NO. 48, 2010 Figure 3. (a) Crystal structure of native monomeric 2m (PDB ID: 1DUZ)28 showing the antiparallel -sheet arrangement of the strands (labeled A to G). (b) Residues that form -strands in ﬁbrillar 2m painted onto the native fold. -Strands in the ﬁbrils10 are shown as thick tubes, and the residues giving rise to assigned intermolecular Ni-CRi cross-peaks are shown in black. The structures were prepared using the Chimera software.29 local dynamics and relaxation whose effects are exacerbated at long mixing times, resulting in large variations in the cross-peak intensities.30 The data presented above suggest that long, straight 2m ﬁbrils grown at pH 2.5 and low salt concentration form parallel, in-register -sheets. In such a case the average distances for intermolecular Ni-CRi and Ni-CRi-1 contacts are ∼5 and ∼5.5 Å respectively (Figure S4), which is consistent with the bulk ZF-TEDOR buildup (Figure S2). In order to accommodate such an arrangement, substantial reorganization of the native antiparallel -sheet structure31-33 is required, indicating that the structure of the monomers within the ﬁbrils must be highly non-native. Figure 3 highlights two clear pieces of evidence for the non-native structure of 2m within ﬁbrils: ﬁrst, residues involved in loops/turns in native 2m (Figure 3a) reorganize to form ordered -strands in the ﬁbrils (Figures 3b and S5), and second, while all -strands form antiparallel -sheet contacts with residues distant in sequence in native 2m, -strands in the ﬁbrils are parallel and in register. The parallel arrangement of the -strands in 2m ﬁbrils was predicted initially by FTIR experiments34,35 and is in agreement with data obtained by site-directed spin labeling and EPR.11 The results described here verify and expand upon the latter, which indicates that spin labels attached to cysteine-substituted residues S33, S55, S61, and T73 among others give EPR spectra indicative of immobile, parallel, and in-register stacked spin labels (Figure S5). Stacks of six 2m monomers arranged in that manner are then required to fulﬁll the electron density maps obtained by cryoEM.12 The site-speciﬁc information regarding the intermolecular arrange- ment of 2m ﬁbrils presented here provides an important step toward a full molecular model of the ﬁbrils. Additional experiments, particularly aimed at determining the quaternary fold of the ﬁbrils, are in progress and should shed light on how this tertiary ﬁbril arrangement ﬁts into such a complex cryoEM electron density proﬁle. Acknowledgment. The authors would like to acknowledge ﬁnancial support from NIH Grants EB003151 and EB002026 and the Wellcome Trust (Grant Numbers 075675 and 062164). We thank Dr. Kendra Frederick for helpful discussions and suggestions. Molecular graphic images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081).