Download Finding the small difference: A nine amino acid extension to

Finding the small difference:
A nine amino acid extension to the hepatitis B core protein
B. Bijttcheri, M.R. Dyson2, R.A. Crowther3
1) Institutfi.irPhysikalische Chemie, Universitgt Freiburg, Albertstral3e23a ,79104 Freiburg, Germany
2) Peptide Therapeutics, 321 Cambridge Science Park, Milton Road, Cambridge CB4 4WG, UK
3) MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
It recently became possible to determine the fold of peptide chains in icosahedrally
organised particles without the necessity of having crystals [ 1,2]. The availability of field emission
gun microscopes equipped with stable cryo stages was one major prerequisite for the collection of
data of sufficient quality. Combination of thousands of particles from micrographs at different
levels of defocus was needed in the subsequent image processing. We describe here how these
methods were employed to locate a nine amino-acid long extension at the N-terminus of hepatitis B
core protein.
Hepatitis B core protein can be expressed in EL coli and forms icosahedrally organised
shells. Predominantly
large shells consisting of 120 dimers are formed, if the C-terminus is
truncated at amino acid 149. Figure 1 shows a micrograph of C-terminally truncated hepatitis B core
protein. Similar shells are formed in a core protein in which the N-terminal two amino acids are
replaced by 11 residues of beta-galactosidase, giving nine additional amino-acids at the N-terminus.
For both species (with and without extension at the N-terminus) independent maps were calculated
to a resolution of 8.5 A. A difference map between the resulting maps was computed to locate the
N-terminal extension. In.figure 2 equatorial slices of the three-dimensional
maps and the difference
map are shown. The equatorial slice gives two almost perpendicular views of the dimers, a narrow
view and a wide view. The slice with the N-terminal extension in figure 2b shows some extra
density at either side of the narrow view of the dimer (marked with arrows), which is missing in the
slice without the N-terminal extension (figure 2a). The difference map shows significant differences
at the same positions (figure 2~).
Figure 3a shows a surface representation of part of the three-dimensional
map of the
core protein without the extension at the N-terminus. The fold suggested for the core protein [I]
places the N-terminus close to local or strict three-fold axes and the C-terminus close to two-fold or
five-fold axes. In figure ‘3b the difference map (map without extension subtracted from map with
extension) is superimposed to the map without extension. All the differences are located around the
strict and local three-fold axes. This is in agreement with the numbering scheme proposed in [l] and
complements the findings in [3], where the C-terminus was located by Undecagold labelling close
to the five-fold and two-fold axes.
From these results it is clear that electron cryo microscopy and image processing of
single icosahedmlly organised particles is a powerful method to detect differences as small as a 9
amino-acid long extension directly without the necessity of extra labelling.
References
1. B. Biittcher et al., Nature, 386 (1997) 88.
2. JP. Conway et al., Nature, 386 (1997) 91.
3. A. Zlotnick et al., Proc. Natl. Acad. Sci. USA, 94 (1997) 9556.
Figure 1 Micrograph of an unstained frozen hydrated sample of hepatitis B core shells (without N-terminal
taken with the Hitachi HF2OOil microscope.
extension),
Figure 2 Equatorial slices through the three-dimensional
map of hepatitis B core she11 without N-terminal extension
from 6154 particles (2a), with N-terminal extension from 3319 particles (2h) and the difference map between both (in
2~). There is some extra density in b (marked with arrows), which is missing in 2a. The difference map in 2c shows a
strong signal at the same position. Negative density differences are shown in grey.
Figure 3 Surface representation
viewed down strict two-fold axis of part of the three-dimensional
map of hepatitis B
core protein without the N-terminal extension. In b the difference between the map with the N-terminal extension and
the map without the extension is overlaid in bright colours on the map shown in a. The difference densities lie around
the strict and local three-fold axes.