Lab Overview
The mission of the Automated Molecular Imaging group is to develop automated
techniques for solving the three-dimensional structure of macromolecular complexes
using cryo electron microscopy (cryoEM). The current focus of the group is
in developing new methods to improve the throughput of the entire process,
from specimen preparation to the generation of the final three-dimensional
map.
Lab Highlight
Evolutionary Optimization of Pressure and Structure in Bacteriophage Lambda
Gabriel Lander and Jack Johnson, TSRI and Alex Evilevitch, Lund University,
Sweden
The double stranded DNA packaged within bacteriophages is highly stressed and
exerts pressures of tens of atmospheres on the capsid walls. It has been hypothesized
that these great pressures aid in ejection of the phage genome into the cell
upon infection as well as play a structural role by doubling the strength of
the capsid wall. We have examined the inter-strand spacing of packaged DNA
within phage lambda for mutants containing 50%, 78%, 94%, and 100% of the wild
type genome by high-throughput cyroEM. We have shown that even a slight change
in the amount of DNA that has been packaged induces a dramatic reorganization
of the inter-strand spacing of the DNA within the phage, suggesting that there
is an evolutionary correlation between genome length and capsid size. It has
also been shown previously that the addition of polyvalent salt ions significantly
decreases the internal pressures within the phage by stabilizing the DNA-DNA-interactions.
Through examination of these specimens by cryoEM, we show that these polyvalent
ions introduce further stabilization by ordering internal DNA that would otherwise
be disordered.
We have additionally begun work towards asymmetric reconstructions of the empty
and fully packaged lambda phage, in which we see an intact three-dimensional
view of the concentrically- spooled DNA within the capsid shell, as well as how
the tail assembly is attached to the capsid. A comparison between central slices
of the asymmetric wild type and empty particles show clear density depicting
the dsDNA and portal proteins,and evidence that upon completion of packaging,
a plug-like gene product or conformational change of the portal shuts the tail
entrance. This unequivocally disproves the theory that there is DNA within the
lambda tail after completion of DNA packaging.
2006 Publications
1. Stagg, S. M., C. Gurkan, D.M. Fowler, P. LaPointe, T.R. Foss, C.S. Potter,
B. Carragher, and W.E. Balch. Structure of the Sec13/31 COPII coat cage. Nature
439, 234-8 (2006).
2. Fellmann, D., Banez, R., Carragher, B. & Potter, C. S. Temperature monitoring
of an EM environment. Microscopy Today, 24-28 (2006).
3. Lander, G. C., Tang, L., Casjens, S. R., Gilcrease, E. B., Prevelige, P.,
Poliakov, A., Potter, C. S., Carragher, B., and Johnson, J. E. (2006). The
structure of an infectious P22 virion shows the signal for headful DNA packaging.
Science 312, 1791-1795.
4. Stagg, S. M., Lander, G. C., Pulokas, J., Fellmann, D., Cheng, A., Quispe,
J. D., Mallick, S. P., Avila, R. M., Carragher, B., and Potter, C. S. (2006).
Automated cryoEM data acquisition and analysis of 284 742 particles of GroEL.
Journal of Structural Biology 155, 470-481.
