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Using Eman to reconstruct asymmetric and/or small proteins

Using Eman to reconstruct asymmetric and/or small proteins. Oscar Llorca. CIB (Centre for Biological Research) Madrid (SPAIN). Contac t at http://electronmicroscopy.cib.csic.es ollorca@cib.csic.es. Asymmetry and small molecules in biology. ~100 kDa. Rho/Rac small GTPase activator

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Using Eman to reconstruct asymmetric and/or small proteins

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  1. Using Eman to reconstruct asymmetric and/or small proteins Oscar Llorca CIB (Centre for Biological Research) Madrid (SPAIN) Contact at http://electronmicroscopy.cib.csic.es ollorca@cib.csic.es

  2. Asymmetry and small molecules in biology ~100 kDa Rho/Rac small GTPase activator (Guanosine Exchange Factor)

  3. SH3 SH2 SH3 CH Ac DH PH ZF Vav3 845 * SH3 SH2 SH3 Ac DH PH ZF Onco-Vav3 144 845 1 class averages Raw particles All data processing performed using Eman Llorca et al., EMBO J. 2005.

  4. FATC FAT FAT N C Catalytic domain HEAT Repeats Ligase IV XRCC4 Ku70 Ku70 DNA-PKcs DNA-PKcs Ku80 Ku80 ARTEMIS p53 PI3-Kinase related family of serine-threonine kinases DNA-PKcs (470 kDa) PI3Kg Helical+kinase HEAT repeats units HEAT repeat (PR65/A subunit) Armadillo repeat (β-catenin) 3649

  5. Electron microscopy field Rivera-Calzada et al., Structure 2005. All data processing performed using Eman

  6. Volume Projection Projection common lines initial reference-free class averages 3D reconstruction Errors in starting volumes

  7. duplications in class averages enlarged volumes generation of false symmetry single particles class average single particles initial model Several conformations of the molecule 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

  8. initial volume Volume Two sides mixed Reference projection Symmetrical 2D average Pseudo symmetry

  9. 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.

  10. CIB, Madrid Angel Rivera-Calzada Ernesto Arias-Palomo Jasminka Boskovic (till end 2004) Institute of Cancer Research, Section of Structural Biology, London David Barford Laurence H. Pearl Centro de Investigación del Cáncer Salamanca Xosé R. Bustelo Contact http://electronmicroscopy.cib.csic.es ollorca@cib.csic.es Breakthrough Breast Cancer Centre, London Clare M. Isacke Sir William Dunn School of Pathology, Oxford Luisa Martinez-Pomares

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