CIB (Centre for Biological Research) Madrid (SPAIN) Contact atOscar Llorca Asymmetry and small molecules in biology ~100 kDa Rho/Rac small GTPase activator (Guanosine Exchange Factor) 1 Vav3 845 CHAcDHPHZF SH3 SH2 Onco-Vav3 845 AcDHPHZF SH3 SH2 144 * Raw particles class averages Llorca et al., EMBO J All data processing performed using Eman PI3-Kinase related family of serine-threonine kinases C FAT FATC Catalytic domain HEAT Repeats N FAT 3649 Armadillo repeat (-catenin) HEAT repeat (PR65/A subunit) HEAT repeats units PI3K Helical+kinase Ku70 Ku80 DNA-PKcs Ku70 Ku80 DNA-PKcs ARTEMIS Ligase IV XRCC4 p53 DNA-PKcs (470 kDa) Electron microscopy field Rivera-Calzada et al., Structure All data processing performed using Eman common lines initial reference-free class averages 3D reconstruction Volume Errors in starting volumes Projection initial model single particles duplications in class averages enlarged volumes generation of false symmetry Initial model is larger than the particles: a- we use a previous reference: (1) negative staining (2) other conformations of the same protein (+- ATP) b- Errors in threed.0a.mrc obtained by common lines single particles class average Several conformations of the molecule projection Symmetrical 2D average Pseudo symmetry Volume initial volume Two sides mixed Reference CONCLUSIONS (a) Angular refinement for small and/or asymmetric molecules still requires development of new protocols and methods. (b) With existing methods, much care must be taken during the building initial volumes and dealing with model bias (c) Up to day, using random conical tilt or any other independent method could be either a good starting point or be used to validate the results. Centro de Investigacin del Cncer Salamanca Xos R. Bustelo ContactInstitute of Cancer Research, Section of Structural Biology, London David Barford Laurence H. Pearl Breakthrough Breast Cancer Centre, London Clare M. Isacke CIB, Madrid Angel Rivera-Calzada Ernesto Arias-Palomo Jasminka Boskovic (till end 2004) Sir William Dunn School of Pathology, Oxford Luisa Martinez-Pomares