velocity methods for the characterization of protein heterogeneity and protein affinity
interactions. Another set of articles describe mass spectrometry (MS)-based approaches
for macromolecular complex analysis. Chapter 10 focuses on the applications of native
mass spectrometry for the characterization of multiprotein complexes ranging from 16 to
801 kDa, Chapter 11 on hydrogen/deuterium exchange mass spectrometry for analyzing
protein–DNA interactions, and Chapter 12 on integrative mass spectrometry-based
approaches for modeling macromolecular assemblies.
Although high‐resolution structure determination using X-ray crystallography or single‐
particle cryo‐electron microscopy (cryo‐EM) is now producing a rapid stream of breakthroughs in structural biology, the preparation of suitable crystals or hydrated frozen
samples on EM grids is often quite challenging. Purified samples, intact and structurally
homogeneous in the test tube, may not crystallize or survive the standard methods of
preparing thin aqueous films on grids. In the case of cryo-EM, optimization of sample
stability and extensive screening of parameters for grid preparation are often required to
collect high-quality datasets. The two last chapters of this section address sample optimization. Chapter 13 provides detailed protocols for preparing negatively stained and hydrated
frozen EM-grid while Chapter 14 addresses solubilization screening using membrane
proteins as model system.
Finally, the third section of this book addresses the characterization of multiprotein
complexes in a cellular environment using state-of-the-art imaging technologies and in vivo
approaches. Chapter 15 describes practical aspects of super-resolution imaging and
Chapter 16 multi-color FRET-FLIM microscopy in live cells. Chapters 17 and 18 present
applications of directed evolution systems and of context-specific and proximity-dependent
labeling using the BioID technology.
This book is expected to be used not only by structural/molecular biologists who need
to prepare multi-components complexes for their own applications but also by scientists
from other fields who are working on macromolecular assemblies from other standpoints
and need an overview of state-of-art approaches. I am especially thankful to all the authors
for their great contributions, devoting their valuable time to the preparation of the manuscripts. I am also indebted to the Series Editor John M. Walker, to the editorial staff
members of Springer and to Marie Christine Poterszman for their kind support in making
this book publishable. I hope that this volume provides a useful overview preparation and
structural analysis of macromolecular complexes and fills a need for well-described hands-on
protocols.
Illkirch, France
Arnaud Poterszman
vi
Preface
interactions. Another set of articles describe mass spectrometry (MS)-based approaches
for macromolecular complex analysis. Chapter 10 focuses on the applications of native
mass spectrometry for the characterization of multiprotein complexes ranging from 16 to
801 kDa, Chapter 11 on hydrogen/deuterium exchange mass spectrometry for analyzing
protein–DNA interactions, and Chapter 12 on integrative mass spectrometry-based
approaches for modeling macromolecular assemblies.
Although high‐resolution structure determination using X-ray crystallography or single‐
particle cryo‐electron microscopy (cryo‐EM) is now producing a rapid stream of breakthroughs in structural biology, the preparation of suitable crystals or hydrated frozen
samples on EM grids is often quite challenging. Purified samples, intact and structurally
homogeneous in the test tube, may not crystallize or survive the standard methods of
preparing thin aqueous films on grids. In the case of cryo-EM, optimization of sample
stability and extensive screening of parameters for grid preparation are often required to
collect high-quality datasets. The two last chapters of this section address sample optimization. Chapter 13 provides detailed protocols for preparing negatively stained and hydrated
frozen EM-grid while Chapter 14 addresses solubilization screening using membrane
proteins as model system.
Finally, the third section of this book addresses the characterization of multiprotein
complexes in a cellular environment using state-of-the-art imaging technologies and in vivo
approaches. Chapter 15 describes practical aspects of super-resolution imaging and
Chapter 16 multi-color FRET-FLIM microscopy in live cells. Chapters 17 and 18 present
applications of directed evolution systems and of context-specific and proximity-dependent
labeling using the BioID technology.
This book is expected to be used not only by structural/molecular biologists who need
to prepare multi-components complexes for their own applications but also by scientists
from other fields who are working on macromolecular assemblies from other standpoints
and need an overview of state-of-art approaches. I am especially thankful to all the authors
for their great contributions, devoting their valuable time to the preparation of the manuscripts. I am also indebted to the Series Editor John M. Walker, to the editorial staff
members of Springer and to Marie Christine Poterszman for their kind support in making
this book publishable. I hope that this volume provides a useful overview preparation and
structural analysis of macromolecular complexes and fills a need for well-described hands-on
protocols.
Illkirch, France
Arnaud Poterszman
vi
Preface