Jose Luis Jimenez

Cryo‐electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing

Amyloid fibrils are assemblies of misfolded proteins and are associated with pathological conditions such as Alzheimers disease and the spongiform encephalopathies. In the amyloid diseases, a diverse group of normally soluble proteins self‐assemble to form insoluble fibrils. X‐ray fibre diffraction studies have shown that the protofilament cores of fibrils formed from the various proteins all contain a cross‐β‐scaffold, with β‐strands perpendicular and β‐sheets parallel to the fibre axis. We have determined the threedimensional structure of an amyloid fibril, formed by the SH3 domain of phosphatidylinositol‐3′‐kinase, using cryo‐electron microscopy and image processing at 25 Å resolution. The structure is a double helix of two protofilament pairs wound around a hollow core, with a helical crossover repeat of ∼600 Å and an axial subunit repeat of ∼27 Å. The native SH3 domain is too compact to fit into the fibril density, and must unfold to adopt a longer, thinner shape in the amyloid form. The 20×40‐Å protofilaments can only accommodate one pair of flat β‐sheets stacked against each other, with very little inter‐strand twist. We propose a model for the polypeptide packing as a basis for understanding the structure of amyloid fibrils in general.

Two Structural Transitions in Membrane Pore Formation by Pneumolysin, the Pore-Forming Toxin of Streptococcus pneumoniae

The human pathogen Streptococcus pneumoniae produces soluble pneumolysin monomers that bind host cell membranes to form ring-shaped, oligomeric pores. We have determined three-dimensional structures of a helical oligomer of pneumolysin and of a membrane-bound ring form by cryo-electron microscopy. Fitting the four domains from the crystal structure of the closely related perfringolysin reveals major domain rotations during pore assembly. Oligomerization results in the expulsion of domain 3 from its original position in the monomer. However, domain 3 reassociates with the other domains in the membrane pore form. The base of domain 4 contacts the bilayer, possibly along with an extension of domain 3. These results reveal a two-stage mechanism for pore formation by the cholesterol-binding toxins.

Structural basis of pore formation by cholesterol-binding toxins

In this paper we describe reconstructions by electron cryo-microscopy of two oligomeric states of the pore-forming toxin pneumolysin. The results are interpreted by the fitting of atomic models of separated domains to the 3-dimensional electron density maps, revealing two steps in the mechanism of pore formation by the family of cholesterol-binding toxins. We briefly describe the observation of the toxin pore in model membranes and contrast the apparent mechanism of pneumolysin with that of other pore-forming toxins.