Download Cells are exposed to DNA damaging agents that can affect their

Using EMAN to obtain 3D reconstructions of asymmetric and/or small proteins
In the recent years, our group has been working on the challenge of using single-particle
EM to build 3D reconstructions of small and/or asymmetric macromolecules (1-5).
These are important steps to solve because most of the molecules of interest in biology
do not have any symmetry, especially those of the so-called “molecular machines”
where a collection of proteins interact to work in such various processes as DNA repair
or RNA splicing.
Also, some important molecules in the size range of 100 kDa are multi-domain proteins
which are difficult to express and crystallize, so that most structural information is
restricted to single domains. Such is the case of many of the molecules implicated in
cell signaling. Therefore, even in the context of relatively small molecules, EM can
provide low resolution 3D reconstructions of the full protein where individual domains
can be fitted.
During these years, our basic processing tool has been the EMAN software, and some
of problems found during image processing will be discussed: asymmetry and pseudosymmetry of the particle, model bias or the presence of a mixture of conformations,
among others.
1- Rivera-Calzada, Maman, J.P., Spagnolo, L., Pearl, L.H. & Llorca O.
Three dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNAPKcs)
Structure (issue February 2005)
2-Boskovic, J., Rivera-Calzada, A, Maman, J.D., Chacón, P., Willison, KR, Pearl, L.H. & Llorca, O.
Visualization of DNA-induced conformational changes in the DNA repair kinase DNA-PKcs
EMBO Journal 22, 5872-5875 (2003)
3-Rivera-Calzada, A., Robertson, D., MacFayden, J., Boskovic, J., Isacke, CM & Llorca, O.
Three dimensional interplay among the ligand binding domains of the uPAR-associated protein Endo180
EMBO reports 4, 807-812 (2003)
4- Llorca, O., Rivera-Calzada, A., Grantham, J. & Willison, K.R.
Electron microscopy and 3D reconsructions reveal that human ATM kinase uses an arm-like domain to
clamp around double-stranded DNA
Oncogene 22, 3867-3874 (2003)
5- Llorca, O., Arias-Palomo, E., Zugaza, J.L. & Bustelo X.R.
Global conformational rearrangements during the physiological and oncogenic activation of the
GDP/GTP exchange factor Vav3
EMBO Journal (in press)