Biology Reference
In-Depth Information
CHAPTER
3
Franck Fourniol
*
,1
, Myl ` ne Perderiset
{
, Anne Houdusse
{
and Carolyn Moores
*
*
Structural Studies of the
Doublecortin Family of MAPs
Institute of Structural and Molecular Biology, Birkbeck College, London, United Kingdom
{
Structural Motility, Institut Curie, Centre National de la Recherche Scientifique, Unite´ Mixte de
Recherche 144, Paris Cedex 05, France
CHAPTER OUTLINE
Introduction .............................................................................................................. 28
3.1 Rationale........................................................................................................... 30
3.2 Methods ............................................................................................................ 32
3.2.1 Expression and Purification of Recombinant Human DCX..................... 32
3.2.1.1 Introduction................................................................................32
3.2.1.2 Expression of DCX ......................................................................33
3.2.1.3 Purification of DCX .....................................................................33
3.2.2 Cryo-ET and Evaluation of DCX-MT Architecture ................................. 37
3.2.2.1 Introduction................................................................................37
3.2.2.2 Sample Preparation ....................................................................37
3.2.2.3 Data Collection ...........................................................................38
3.2.2.4 Structure Determination..............................................................38
3.2.3 Cryo-EM and Subnanometer Resolution 3D Structure Determination..... 39
3.2.3.1 Introduction................................................................................39
3.2.3.2 Sample Preparation and Data Collection......................................39
3.2.3.3 Setup and Automated Processing ...............................................39
3.2.3.4 Initial 3D Model and Reference Projections .................................39
3.2.3.5 Finding the Seam and Image Selection........................................39
3.2.3.6 Reconstructions of DCX-K-MTs ..................................................40
3.2.3.7 Pseudo-atomic Model Building....................................................40
3.2.3.8 Insight into MT Structure in the Absence of Stabilizing Drugs.......42
1
Current address: Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories,
44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.
