title: High-resolution single-particle 3D analysis on GroEL prepared by cryo-negative staining.
abstract: Cryo-negative staining was developed as a complementary technique to conventional cryo-electron microscopy on supramolecular complexes. It allows imaging biological samples in a comparable state of structural preservation to conventional cryo-EM but the staining produces better contrast in accessible areas and allows data recording at lower defocus values. Cryo-negative staining vitrifies biological particles in the presence of a concentrated ammonium molybdate solution at neutral pH. It was successfully used to study the structure and dynamics of several macromolecules, such as human transcription factors and RNA polymerases. Imaging macromolecular complexes with cryo-negative staining has been established previously to better than 2 nm detail. However, it has not been verified so far whether cryo-negative staining also visualizes secondary structure elements. Using the well known E. coli GroEL chaperonin, we could show that the structure is well preserved to approximately 10 A resolution. Secondary structure details are at least partially resolved in the electron density map.
authors: Molecular, Cellular and Developmental Biology Department, Campus Box 347, University of Colorado at Boulder, Boulder, CO 80309-0347, USA. sacha@bio3d.colorado.edu
authors: Sacha
authors: S
authors: De Carlo
authors: Nicolas
authors: N
authors: Boisset
authors: Andreas
authors: A
authors: Hoenger
mesh_terms: D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry; D016624:Negative Staining / Q000379:methods*; D020836:Protein Structure, Quaternary
chemical_list: D018834:Chaperonin 60; D046912:Multiprotein Complexes