Electron Cryomicroscopy of Fibrillar Collagens - Methods in Molecular Biology: Extracellular Matrix Protocols - page 90

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Electron Cryomicroscopy of Fibrillar Collagens
Roger S. Meadows, David F. Holmes, Chris J. Gilpin,
and Karl E. Kadler
1. Introduction
Collagen fibrils in tissue are generally heterotypic with more than one type
of collagen molecule incorporated into the fibril structure. Furthermore spe-
cific macromolecules are bound onto the fibril surface influencing both the
assembly and the interaction of the fibril with the surrounding matrix. The
electron cryomicroscopy procedures described here form part of a program of
work to determine the structure and assembly of tissue fibrils containing sur-
face-associated components.
Collagen fibrils show a characteristic axial periodicity of ~67 nm, which is
most apparent in the gap/overlap structure of fibrils that have been prepared
for transmission electron microscopy by negative staining. The details of this
pattern can be related to the axial distribution of amino acids in the collagen
triple helix when each residue is scored for “bulkiness” (1) . The negative stain
distribution does not, however, allow the scattering mass of the proteins to be
determined nor is it possible to distinguish between contributions from internal
and surface structure in single projections. We have used dark-field scanning
transmission electron microscopy (STEM) to mass-map the unstained collagen
fibril allowing the mass per unit length to be measured along the fibril axis (2) .
In collagen fibrils made in vitro from type I collagen (in the absence of other macro-
molecule), it is possible to determine the number of collagen molecules in the
fibril cross-section (3) . Studies of the axial mass distribution by STEM within
a single D -period are limited, however, by differential shrinkage brought about
by dehydration of the fibrils (2) . Consequently, fine structure below 10 nm is lost.
Two techniques to overcome these problems are detailed in this chapter.
The first is electron cryomicroscopy of unstained collagen fibrils in vitreous