Because of the large size of the HSV-1 capsid, any overall particle
deformation and the defocus gradient across the particle (that is, the Ewald
sphere effect) (73 (link)) would result in
considerable dampening of the high-resolution information in conventional
icosahedral reconstruction by treating the particle as a whole. Both effects are
the most severe in the capsid vertex region, limiting attainable resolution of
the penton and the CATC. To further improve resolution of the vertex region, we
applied a subparticle refinement and reconstruction procedure considering both
local variations (74 (link)) and defocus
gradient. Specifically, icosahedral orientation and center parameters of each
particle image determined above were used to guide extraction of all vertex
regions as subparticles with their defocus values adjusted according to their
locations on each particle. The orientation and center parameters of these
sub-particles were locally refined with Relion (75 (link)), and, by imposing C5 symmetry, we obtained a final
reconstruction at 3.5-Å resolution on the basis of the gold-standard FSC
= 0.143 criterion (68 (link)). Atomic
models of components in the vertex region (including hexon MCPs P1, P2, and P6;
penton MCP; triplex Ta; and the CATC) were fitted into the refined subparticle
map, manually checked in Coot, and refined with Phenix. Overall, our models
built from the 4.2-Å resolution icosahedral map match well with the
improved subparticle map at 3.5-Å resolution, indicative of good quality
and validity of our original models.
deformation and the defocus gradient across the particle (that is, the Ewald
sphere effect) (73 (link)) would result in
considerable dampening of the high-resolution information in conventional
icosahedral reconstruction by treating the particle as a whole. Both effects are
the most severe in the capsid vertex region, limiting attainable resolution of
the penton and the CATC. To further improve resolution of the vertex region, we
applied a subparticle refinement and reconstruction procedure considering both
local variations (74 (link)) and defocus
gradient. Specifically, icosahedral orientation and center parameters of each
particle image determined above were used to guide extraction of all vertex
regions as subparticles with their defocus values adjusted according to their
locations on each particle. The orientation and center parameters of these
sub-particles were locally refined with Relion (75 (link)), and, by imposing C5 symmetry, we obtained a final
reconstruction at 3.5-Å resolution on the basis of the gold-standard FSC
= 0.143 criterion (68 (link)). Atomic
models of components in the vertex region (including hexon MCPs P1, P2, and P6;
penton MCP; triplex Ta; and the CATC) were fitted into the refined subparticle
map, manually checked in Coot, and refined with Phenix. Overall, our models
built from the 4.2-Å resolution icosahedral map match well with the
improved subparticle map at 3.5-Å resolution, indicative of good quality
and validity of our original models.
