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are bigger than
∆
N25 and
∆
C327. The quantity of
∆
C335 VLPs was
80% of full-length VLPs, while the amount of
C327 VLPs sharply
decreased to 4.5%. The result indicates that the 328-335 residues play
a crucial role in assembling VLPs. The relative percentages of VLPs
from
∆
C335 indicate
that the ability to form VLPs declined as successive residues were
removed. Particularly, deleting four residues at 335-338 (
∆
C327,
∆
C328,
∆
C330,
∆
C333,
∆
C334, and
∆
C334)
significantly reduced the quantity of VLPs to 39.4%, while removing
three residues at 336-338 (
∆
C335) only reduced the percentage to
80%. An excess of deleting one residue at 335th (Asp-335) decreases
VLPs to 39.4% from 80% of full-length capsid protein.
∆
Electron Cryomicroscopy and Image Reconstruction
In order to obtain higher-resolution structural information about the
fish virus VLPs, particles were flash-frozen for analysis by low-dose
electron cryomicroscopy.
5
A total of 263 particles were used to gen-
erate a three-dimensional reconstruction at 23 Å resolution (Fig. 1).
The particle had a diameter of 380 Å and the morphology was con-
sistent with the expected
T
3 surface lattice previously observed for
insect nodaviruses. As in the insect viruses, prominent protrusions
were clearly visible at the quasi-threefold symmetry axes, but overall
the surface features were strikingly different. For example, the pro-
trusion at the quasi-threefold axis was significantly larger than that
found in the insect viruses Flock house virus and Pariacoto virus
=
Fig. 1.
CryoEM maps of MGNNV at 23 Å resolution.
(Reprinted from Tang
et al.
, 2002.)
