Supporting InformationWille et al. 10.1073/pnas.0909006106SI MethodsPreparation of PrP 27–30. SHaPrP 27–30, strain Sc237: N-terminally truncated PrP 27–30 was prepared from the brains ofSyrian hamsters infected with Sc237 prions. The purification wasbased on multiple precipitation and detergent extraction steps(1). During the purification procedure PrPSc was truncated bylimited digestion with proteinase K (PK). The purified prionsuspension was pelleted by centrifugation (100,000 � g for 1 h)and resuspended in concentrated form in water. More than 100hamster brains were required to produce enough PrP 27–30 forone or two fiber diffraction samples.

MoPrP 27–30, strain RML: N-terminally truncated PrP 27–30was prepared from the brains of RML prion-infected FVB miceexhibiting signs of neurological dysfunction. The purification wasbased on the specific precipitation of PrP 27–30 by PTA (2).Before the PTA precipitation, PrPSc was truncated by limiteddigestion with PK in the presence of 2% (wt/vol) Sarkosyl. ThePTA-precipitated PrP 27–30 was further purified by centrifuga-tion through a cushion of 50% sucrose in the presence of 0.2%Sarkosyl. The resulting pellet was resuspended in buffer con-taining 0.2% Sarkosyl. Finally, the purified prion preparationwas pelleted by centrifugation (100,000 � g for 1 h) andresuspended in concentrated form in water. The substantiallyhigher yield of this preparation technique reduced the require-ment for one or two diffraction samples to about 20 mousebrains.

MoSP1 ‘‘synthetic prions’’ were prepared from the brains ofTg9949 mice overexpressing N-terminally truncated Mo-PrP(�23–88), infected with MoSP1 synthetic prions. No prote-olysis step was necessary as these mice express a truncatedMoPrPSc that is equivalent to PrP 27–30. The purification wasperformed as described for MoPrP 27–30(RML).

The procedures that were used to purify PrP 27–30 did notaffect its infectivity. Bioassays of the concentrated PrP 27–30samples that were used for preparing fiber diffraction specimensroutinely indicated prion titers in excess of about 8 log ID50/mL,which is probably an underestimate due to the saturation limit ofthe animal model. On several occasions, PrP 27–30 dried fibersthat had been exposed to the focused synchrotron x-ray beamwere bioassayed. Even after exposure of the samples to theintense synchrotron radiation the remaining prion titers wereoften above 6 log ID50/mL.

Preparation of Recombinant PrP Amyloid. RecMoPrP (89–230) andrecSHaPrP (90–231) amyloids were prepared from murine PrP(89–230) and Syrian hamster PrP (90–231), respectively, ex-pressed in E. coli. Recombinant PrP was unfolded by denatur-ation in 10 M urea. Upon dilution to 5 M urea, oligomers richin �-structure (�-oligomers) were formed; further dilution intosaline induced formation of amyloid fibers (3). In bioassays,these amyloid preparations demonstrated prion infectivity (4).For the fiber diffraction experiments, the amyloid fibers werepelleted from their buffer solution by centrifugation (100,000 �g for 1 h) and resuspended in a concentrated form in water.

Fiber Diffraction Sample Preparation. Fiber diffraction sampleswere made by suspending a drop of protein solution (typically 5to 10 �L, containing 50 to 100 �g purified prions) between twoglass rods in a closed chamber (5), with saturated potassiumsulfate solution producing 97% relative humidity. Silicon nitridewindows allowed transmission of X-rays, while maintaininghumidity and serving as primary containment.

X-Ray Diffraction Data Collection. X-ray fiber diffraction dataincluding data shown in Fig. 1 were collected at the BiologicalSmall-Angle X-ray Scattering beamline 4–2 at Stanford Syn-chrotron Radiation Laboratory (6). For Fig. 1 A, the beamdiameter was approximately 200 �m, data were recorded usinga MarCCD 165 detector (raster size 79 �m) approximately 228mm from the specimen, and the X-ray wavelength was 1.109 Å.For Fig. 1B, the beam diameter was approximately 100 �m, datawere recorded using a Rayonix MX225-HE detector (raster size73 �m) approximately 325 mm from the specimen, and the X-raywavelength was 1.078 Å. Data in Fig. 2 were collected at theBioCARS beamline at the Advanced Photon Source at ArgonneNational Laboratory, and later confirmed by collecting datafrom similar, freshly made fibers at SSRL. The beam diameterwas approximately 150 �m. Data were recorded using an ADSCQuantum-315 detector (raster size 103 �m) approximately 250mm from the specimen. The X-ray wavelength was 0.9 Å.

Fiber Diffraction Data Analysis. Diffraction patterns were analyzedusing the program WCEN (7). Specimen to detector distanceswere determined by measuring diffraction patterns from tobaccomosaic virus (TMV), using the known helical pitch of TMV (8)as a standard for calibration.

Model Construction and Diffraction Simulation. Molecular modelswere constructed using SwissPDBViewer (9). Diffraction simu-lations were carried out using the programs FNV and DISOR-DER, from the FiberNet website (www.fiberdiffraction.org).Both programs perform Fourier-Bessel transforms to calculatediffraction from helical structures. DISORDER also simulatesorientational disorder. Display of diffraction patterns used theprograms FIT2D (www.esrf.eu/computing/scientific/FIT2D/),WCEN (7), and FibreFix (10). An isotropic temperature factorof 10 was used in all simulations unless otherwise indicated.

Electron Microscopy. For electron microscopy (11), formvar/carbon-coated 200 mesh copper grids (Ted Pella, Inc.) were glowdischarged before negative staining. Five-�L samples were ad-sorbed for approximately 30 s, then the grids were washed with1 to 3 drops (50 �L) each of 0.1 M and 0.01 M ammonium acetate(pH 7.4); the grids were then stained with 2 drops freshly filtered2% ammonium molybdate or 2% uranyl acetate. After drying,the samples were viewed in a FEI Tecnai F20 electron micro-scope at 80 kV and a standard magnification of 25,000. Electronmicrographs were recorded with a Gatan Ultrascan CCD cam-era. The magnification was calibrated using negatively stainedcatalase crystals and ferritin. Widths of the filaments weremeasured on electron micrographs taken at a higher magnifi-cation (80,000). The measurements were made by determiningthe minimal width of the stain-excluding density of clearlyrecognizable filaments.

Fourier Transform Infrared Spectroscopy (FTIR). One-�L aliquots ofsamples used for fiber diffraction were dried on a 0.5-mm AgClwindow as discrete spots. After complete drying in N2, extinctionspectra were recorded with AgCl as a reference. Trace amountsof detergents and salts did not interfere with the amide Iabsorption band (1,700–1,600 cm�1). On a Perkin-Elmer System2000 FTIR spectrometer, 64 double-sided interferograms werescanned bidirectionally and averaged for each sample. ThePerkin-Elmer standard microscope attachment, equipped withan MCT detector and purged with dry N2, was used to limit the

Wille et al. www.pnas.org/cgi/content/short/0909006106 1 of 8

illuminated sample spot. Spectra were recorded with 2 cm�1

instrument resolution, and 4-fold zero-filling yielded one datapoint per 0.5 cm�1. Trace signals from water vapor wereeliminated with the suppression algorithm of the SpectrumGXV5 software (Perkin-Elmer). Low frequency noise was filteredwith a Fourier-self deconvolution algorithm at 6 cm�1 cut-off,and the amide I band was baseline corrected. Secondary struc-ture information was derived from standardized amide I banddecomposition (12). In brief, Cauchy curves were summed toapproximate the shape of the amide I band. The Cauchy curveintegrals were used to calculate the fraction of secondarystructure assigned to the peak frequency of the curve. Fitting theamide I bands of all samples with identical initialization param-eters for the number of bands, band position, width, shape, andthe automatic determination of each intensity as well as theoverall baseline values resulted in a high sensitivity for structuralalterations. Because of the diversity of samples, a calibration (12)was not applicable. The absolute values for the secondarystructure fractions are estimates with an approximately � 10%variation.

SI ACKNOWLEDGMENTS. Use of the Advanced Photon Source was supportedby the U.S. Department of Energy, Basic Energy Sciences, Office of Science,under contracts W-31-109-ENG-38 and DE-AC02-06CH11357. Use of the Bio-CARS Sector 14 was supported by the National Institutes of Health, NationalCenter for Research Resources, under grant number RR007707. BioCAT is aNational Institutes of Health-supported Research Center RR-08630. The con-tent is solely the responsibility of the authors and does not necessarily reflectthe official views of the National Center for Research Resources or the Na-tional Institutes of Health.

Portions of this research were carried out at the Stanford SynchrotronRadiation Laboratory, a national user facility operated by Stanford Universityon behalf of the U.S. Department of Energy (DOE), Office of Basic EnergySciences. The SSRL Structural Molecular Biology Program is supported by theDOE, Office of Biological and Environmental Research, and by the NationalInstitutes of Health, National Center for Research Resources, Biomedical Tech-nology Program.

X-ray scattering and diffraction technologies and their applications to thedetermination of macromolecular shapes and conformations at the SIBYLSbeamline at the Advanced Light Source, Lawrence Berkeley National Labora-tory, are supported in part by the DOE program Integrated Diffraction Anal-ysis Technologies (IDAT) and the DOE program Molecular Assemblies Genesand Genomics Integrated Efficiently (MAGGIE) under Contract Number DE-AC02–05CH11231 with the U.S. DOE.

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2. Safar J, et al. (1998) Eight prion strains have PrP(Sc) molecules with different confor-mations. Nature Med 4:1157–1165.

3. Baskakov IV, Legname G, Baldwin MA, Prusiner SB, Cohen FE (2002) Pathwaycomplexity of prion protein assembly into amyloid. J Biol Chem 277:21140 –21148.

4. Legname G, et al. (2004) Synthetic mammalians prions. Science 305:673–676.5. McDonald M, Kendall A, Tanaka M, Weissman JS, Stubbs G (2008) Enclosed chambers

for humidity control and sample containment in fiber diffraction. J Appl Crystallogr41:206–209.

6. Smolsky IL, et al. (2007) Biological small-angle X-ray scattering facility at the StanfordSynchrotron Radiation Laboratory. J Appl Cryststallogr 40:s453–s458.

7. Bian W, Wang H, McCullough I, Stubbs G (2006) WCEN: A computer program for initialprocessing of fiber diffraction. J Appl Crystallogr 39:752–756.

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10. Rajkumar G, AL-Khayat HA, Eakins F, Knupp C, Squire JM (2007) The CCP13 FibreFixprogram suite: Semi-automated analysis of diffraction patterns from non-crystallinematerials. J Appl Crystallogr 40:178–184.

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Fig. S1. Diffraction pattern from recMoPrP (89–230) amyloid. Color table (7) used for this figure is shown.

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Fig. S2. SDS/PAGE analysis of prion samples used in fiber diffraction experiments. Following SDS/PAGE, gels were either silver-stained (Ag) or immunoblottedusing anti-PrP monoclonal antibody P (13) (WB), as labeled. Lanes 1 and 2 were loaded with PrP 27–30 purified from the brains of scrapie-sick FVB mice infectedwith RML prions. The purification procedure was based on limited proteolysis with PK and specific precipitation with PTA (2). This sample gave the diffractionpattern shown in Fig. 2A and was also used for the negative stain electron microscopy shown in Fig. 4C. A reasonable level of purity was achieved, but in thesilver stained gel, a few minor contaminating bands were still visible. Lanes 3 and 4 were loaded with PrP 27–30 purified from the brains of scrapie-sick Tg9949mice infected with MoSP1 prions. The purification relied solely on the specific precipitation with PTA—no PK digestion was applied. This sample gave thediffraction pattern shown in Fig. 2B and was also used for the negative stain electron microscopy shown in Fig. 4E. Here, PrP 27–30 was only a minor fractionof the sample and many additional protein bands dominated the silver stained gel, illustrating the beneficial effects of the limited proteolysis step.

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Fig. S3. Helical models for amyloid structure and their calculated diffraction patterns. (A) Water-filled nanotube protofilament model (14), viewed down thehelical axis and perpendicular to the axis. (B) Calculated diffraction pattern from a single water-filled nanotube protofilament. (C) Cross-section of a trimericwater-filled nanotube model. (D) Calculated diffraction pattern from C. (E) �-helical model for PrP 27–30 (15) viewed down the helical axis and perpendicularto the axis. (F) Calculated diffraction pattern from a single PrP 27–30 �-helical protofilament. (G) F with a large temperature factor (5,000) applied to theC-terminal �-helical domain. (H) F with the repeating unit of two monomers arranged head to head. (I) H with a large temperature factor (5,000) applied to theC-terminal �-helical domain. (J) Cross-section of a trimeric �-helical model for PrP 27–30. (K) Calculated diffraction pattern from a trimeric �-helical PrP 27–30fibril. (L) K with a large temperature factor (5,000) applied to the C-terminal �-helical domain. (M) K with the repeating unit of two trimers arranged head tohead. (N) M with a large temperature factor (5,000) applied to the C-terminal �-helical domain. (O–T) Calculated diffraction patterns from trimeric PrP 27–30�-helical filaments with short crossover lengths (200 Å–400 Å) and a short coherent length (50 Å). (O) Stack of 10 slightly disordered trimers; the three monomersin a trimer are not in an exact 3-fold symmetry, but are tilted a few degrees from ideal positions. (P) Filament with an azimuthal rotation between consecutivetrimers of 36°. (Q) Filament with an azimuthal rotation of 18°. (R) Filament with an azimuthal rotation of 18°, with each monomer in the trimer tilted 15° abouta radius. (S) R with a large temperature factor (5,000) applied to the C-terminal �-helical domain. (T) S with each monomer slightly disordered by randomdisplacement of about 1 Å, an isotropic temperature factor of 80 applied to the �-helical domain, and 5,000 applied to the C-terminal �-helical domain. Thetemperature factor of 5,000 applied to the C-terminal domain in some cases is somewhat arbitrary, but it is included to represent the possibility that this domainmay be highly disordered, as the models suggest it may be.

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Fig. S4. Stacked �-sheet models for amyloid structure. Each panel shows the cross-section of a model with its calculated diffraction pattern. The cross-sectionsconsist of two to eight peptide pairs in various arrangements. The peptide pair structure is taken from the crystal structure of the amyloidogenic peptideGNNQQNY (16).

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Fig. S5. Color tables (7) used for Figs. 1 and 2. (A) Fig. 1A (inset). (B) Fig. 1A. (C) Fig. 1B. (D) Fig. 2A. (E) Fig. 2A (inset). (F) Fig. 2B.

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Table S1. Fourier transform infrared spectroscopic characterization of fiber diffraction samples

Sample �-helix �-sheet Turn Coil

SHa Sc237 PrP 27–30 36% 42% 8% 13%FVB RML PrP 27–30 21% 39% 4% 35%recMoPrP(89–230) amyloid 35% 40% 12% 13%

Aliquots of the samples that were used for fiber diffraction experiments were also subjected to characterization by FTIR. All samples contained predominantly�-sheet structure, but retained a substantial portion of �-helix.

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