Using Eman to reconstruct asymmetric and/or small proteins

CIB (Centre for Biological Research)Madrid (SPAIN)

Contact at

 http://electronmicroscopy.cib.csic.es

ollorca@cib.csic.es

Oscar Llorca

Asymmetry and small molecules in biology

~100 kDaRho/Rac small GTPase activator

(Guanosine Exchange Factor)

1Vav3

845CH Ac DH PH ZF SH3 SH3SH2

Onco-Vav3845

Ac DH PH ZF SH3 SH3SH2144

*

Rawparticles

classaverages

Llorca et al., EMBO J. 2005.

All data processing performed using Eman

PI3-Kinase related family of serine-threonine kinases

CFAT

FATC

Catalytic domain

HEAT Repeats

N FAT

3649Armadillo repeat

(β-catenin)HEAT repeat

(PR65/A subunit)

HEAT repeats units PI3KHelical+kinase

Ku70Ku80

DNA-PKcsKu70Ku80

DNA-PKcs

ARTEMIS

Ligase IVXRCC4

p53

DNA-PKcs (470 kDa)

Electron microscopy field

Rivera-Calzada et al.,Structure 2005. All data processing performed using Eman

common lines

initial reference-freeclass averages

3D reconstruction

Volume

Errors in starting volumes

Projection

Projection

initial model single particles

duplications in class averagesenlarged volumesgeneration 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 conformationsof 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 Investigación del CáncerSalamanca

Xosé R. Bustelo

Contact

 http://electronmicroscopy.cib.csic.es

ollorca@cib.csic.es

Institute of Cancer Research, Section of Structural Biology, London

David BarfordLaurence H. Pearl

Breakthrough Breast Cancer Centre, London

Clare M. Isacke

CIB, Madrid

Angel Rivera-CalzadaErnesto Arias-Palomo

Jasminka Boskovic (till end 2004)

Sir William Dunn School of Pathology, Oxford

Luisa Martinez-Pomares