using eman to reconstruct asymmetric and/or small proteins
Post on 03-Feb-2016
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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
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