1
Supplementary Information
Figure S1 The V=0.5 Harker section of an anomalous difference Patterson map
calculated using diffraction data from the NNQQNY crystal at 1.3 Å resolution.
The position of the single zinc site can be inferred from the presence of 5 
peaks. The map is contoured at 1  intervals starting at 1 . Anomalous
difference coefficients were calculated with the program XPREP (Bruker,
Madison, WI). The difference Patterson map was calculated and displayed
using the XtalView1 suite of programs.
2
Figure S2 The cross beta diffraction pattern characteristic of GNNQQNY fibrils
is also characteristic of GNNQQNY single crystals. The intensities from the
single crystal are mapped onto the page to simulate the rotational disorder
found in the fibril sample. Specifically, the disorder is modelled as a complete
rotation about the crystal b axis (or fibril axis, vertical in this figure) coupled with
a 10 degree tilt. The 4.7 Å meridional band can be attributed to the crystal's
011 reflection and the 11.3 Å equatorial band can be attributed to the 002
reflection. Similarities can also be found in the intensities and positions of
additional meridional bands.
3
Figure S3 The structure of NNQQNY. Unless otherwise noted, carbon atoms
are coloured in grey/white, nitrogen in blue, oxygen in red, and water molecules
are denoted by red crosses. a, A view along the b axis of the NNQQNY crystal,
similar to the view of GNNQQNY shown in Figure 2c. The packing of side-
4
chains in the dry and wet interfaces is nearly identical between the two peptide
crystals. These interfaces are highlighted with shaded ovals: grey for dry and
red for wet. The main difference between the two peptide structures is the
presence of Zn2+ ions (yellow spheres), which link the N- and C-termini of
neighbouring NNQQNY peptide chains. Acetate anions (removed for clarity)
serve as the other ligands to zinc and balance its positive charge. b,
Coordination of Zn2+. Each zinc cation is coordinated by two acetate anions, a
carboxyl-terminal oxygen, and an amino-terminal nitrogen. The nitrogen is
therefore uncharged (i.e. -NH2), using a lone pair to coordinate the zinc. The
coordination geometry is roughly tetrahedral. Black dots show coordination of
the zinc, and numbers show the coordination distances in Ångstroms. c, The
peptide-peptide contact of the wet interface is a tyrosine stack. This
arrangement of tyrosines is also seen in the GNNQQNY structure. Side chain
carbon atoms of tyrosine are coloured in green. d,e, Hydrogen bonding within a
sheet as seen from the dry (d) and wet (e) interfaces. The organization of side
chain and backbone hydrogen bonds within a sheet is the same as that of
GNNQQNY. Side chain carbons are shown in green. Side chain-side chain
hydrogen bonds are traced by green dots, while backbone-backbone and
backbone-sidechain hydrogen bonds are traced with black/grey dots.
5
Table S1 Dihedral angles* in the structure of GNNQQNY
Residue

Gly1


-162
171



Asn2
-61
141
-176
-57
-60
Asn3
-119
125
166
-176
61
Gln4
-126
112
173
-164
-160
54
Gln5
-115
126
-176
-62
159
-72
Asn6
-116
97
-171
-54
66
Tyr7
-80
-60
91
*Parallel -sheets have average values of = -119, =113.
6
Structure-based energetics
With several assumptions, we can make a crude estimate of the free energy barrier to
formation of the pair-of-sheets structure. We assume that a nucleus consists of 3-4
GNNQQNY molecules, two in one sheet interdigitated with 1-2 in the opposing sheet.
This unit can act as a template for the addition of other GNNQQNY molecules. We
assume the transition state structure is one molecule short of the nucleus. The standard
free energy change for incorporation of a GNNQQNY molecule into the growing sheet,
ΔG0 = ΔH0 - T ΔS0, has both enthalpic and entropic components, the latter accounting
for the barrier to forming this highly organized structure. One component to the entropy
is the decrease of translational and rotational entropy that accompanies the
incorporation of one GNNQQNY molecule into a protofibril. This can be estimated by
the method of Erickson2 as about -21 cal/mol-K. To this must be added the entropy
change for the GNNQQNY backbone to acquire the extended conformation of a
strand, which we can estimate from Boltzmann’s formula: S=-Rlnwhere R is the
gas constant and is the number of configurations of GNNQQNY that are lost when it
enters the sheet. This entropy is roughly -R ln 26 = -8 cal/mol-K, because there are 6
residues that participate in the sheet (the initial Gly does not), and these residues can be
in either the  or  conformation. We can also use Boltzmann’s formula to estimate the
entropy loss as the Gln and two Asn side chains acquire their proper rotamers in the dry
interface. Because the Gln side chain has free rotation about three single bonds, each
with three low energy conformations, and the two Asn side chains each have rotations
about two bonds, the entropy change is –R ln(33x32x32) = -15 cal/mol-K. The sum of
these three entropy decreases is -44 cal/mol-K or a free energy increase of ~13
kcal/mole-of-GNNQQNY at 300 K. This decrease of entropy is partially compensated
by an increase of entropy caused by the hydrophobic effect upon cross- spine
formation. This increase can be crudely estimated by the amount of apolar accessible
surface area buried3,4 as each GNNQQNY molecule enters the pair-of-sheets structure,
7
which is 249 Å2, corresponding to roughly -5 kcal/mol at 300 K. Thus the free energy
barrier from entropy effects is ~8kcal/mol-of-GNNQQNY at room temperature. If there
are three molecules in the transition state complex, the barrier is ~24 kcal/mole, a
substantial barrier to fibril formation.
The minimum nucleus size for fibril formation of full length Sup35 has been
found by Collins et al.5 to be a monomer (or possibly a dimer or trimer). One way of
reconciling this finding with the somewhat larger nucleus inferred here for GNNQQNY
is that several segments of a monomer of the full protein participate to form an
intramolecular nucleus. Consistent with this, EPR studies of Tanaka et al.6 reveal that
an extensive region of the Sup35 prion domain becomes highly ordered upon fibril
formation.
References:
1.
McRee, D. E. XtalView/Xfit--A versatile program for manipulating atomic
coordinates and electron density. J Struct Biol 125, 156-65 (1999).
2.
Erickson, H. P. Co-operativity in protein-protein association. The structure and
stability of the actin filament. J Mol Biol 206, 465-74 (1989).
3.
Eisenberg, D. & McLachlan, A. D. Solvation energy in protein folding and
binding. Nature 319, 199-203 (1986).
4.
Eisenberg, D., Wesson, M., Yamashita, M. Interpretation of Protein Folding and
Binding with Atomic Solvation Parameters. Chemica Scripta 29A, 217-221
(1989).
5.
Collins, S. R., Douglass, A., Vale, R. D. & Weissman, J. S. Mechanism of prion
propagation: amyloid growth occurs by monomer addition. PLoS Biol 2, e321
(2004).
6.
Tanaka, M., Chien, P., Naber, N., Cooke, R. & Weissman, J. S. Conformational
variations in an infectious protein determine prion strain differences. Nature
428, 323-8 (2004).