Sticholysin II (StnII) is a pore-forming protein (PFP) produced by the sea anemone Stichodactyla helianthus. We found out that StnII exists in a monomeric soluble state but forms tetramers in the presence of a lipidic interface. Both structures have been independently determined at 1.7 A and 18 A resolution, respectively, by using X-ray crystallography and electron microscopy of two-dimensional crystals. Besides, the structure of soluble StnII complexed with phosphocholine, determined at 2.4 A resolution, reveals a phospholipid headgroup binding site, which is located in a region with an unusually high abundance of aromatic residues. Fitting of the atomic model into the electron microscopy density envelope suggests that while the beta sandwich structure of the protein remains intact upon oligomerization, the N-terminal region and a flexible and highly basic loop undergo significant conformational changes. These results provide the structural basis for the membrane recognition step of actinoporins and unexpected insights into the oligomerization step.

The influenza viruses cause annual epidemics of respiratory disease and occasional pandemics, which constitute a major public-health issue. The segmented negative-stranded RNAs are associated with the polymerase complex and nucleoprotein (NP), forming ribonucleoproteins (RNPs), which are responsible for virus transcription and replication. We describe the structure of native RNPs derived from virions. They show a double-helical conformation in which two NP strands of opposite polarity are associated with each other along the helix. Both strands are connected by a short loop at one end of the particle and interact with the polymerase complex at the other end. This structure will be relevant for unraveling the mechanisms of nuclear import of parental virus RNPs, their transcription and replication, and the encapsidation of progeny RNPs into virions.

Three-dimensional reconstruction from cryoelectron micrographs of the eukaryotic cytosolic chaperonin CCT complexed to tubulin shows that CCT interacts with tubulin (both the a and b isoforms) using ®ve speci®c CCT subunits. The CCT±tubulin interaction has a different geometry to the CCT±actin interaction, and a mixture of shared and unique CCT subunits is used in binding the two substrates. Docking of the atomic structures of both actin and tubulin to their CCT-bound conformation suggests a common mode of chaperonin±substrate interaction. CCT stabilizes quasi-native structures in both proteins that are open through their domain-connecting hinge regions, suggesting a novel mechanism and function of CCT in assisted protein folding. Keywords: actin/chaperonin/electron microscopy/protein folding/tubulin IntroductionFolding of many proteins in vivo requires interaction with macromolecular complexes known as chaperonins. These proteins are ubiquitous oligomeric assemblies that have been classi®ed into two distinct families that share limited but signi®cant sequence homology: type I, present in eubacteria and endosymbiotic organelles, and of which the bacterial GroEL is the best known representative; and type II, present in archaebacteria and the eukaryotic cytosol, which are represented by the thermosome and CCT (chaperonin containing TCP-1), respectively (Bukau and Horwich, 1998;Gutsche et al., 1999;Willison, 1999). Most of the chaperonins share a common architecture, a cylinder made up of two back-to-back stacked rings, each one enclosing a cavity where folding takes place. The atomic structures of GroEL (Braig et al., 1994) and the type II thermosome (Ditzel et al., 1998) have revealed a common subunit architecture consisting of three domains: apical, intermediate and equatorial. The equatorial domain provides most of the intra-and inter-ring interactions and contains the binding site for ATP, the hydrolysis of which is necessary for the working cycle of the chaperonin, while the apical domain is involved in substrate binding and undergoes large conformational changes during the folding cycle. There are, however, numerous differences between type I and type II chaperonins, one of which is the absence of co-chaperonins for type II family members, whose role in the closure of the cavity during the chaperonin working cycle is ful®lled instead by a helical protrusion in the apical domain (Klumpp et al., 1997;Ditzel et al., 1998;Llorca et al., 1999a). Another important difference is related to the degree of complexity of the chaperonin ring, ranging from the seven identical subunits of type I chaperonin GroEL to eight different polypeptide subunits in the case of the type II chaperonin CCT. The most important difference between these two chaperonins is, however, related to their substrate speci®city: whereas GroEL interacts with a broad range of substrates (Houry et al., 1999) using a non-speci®c recognition mechanism based on hydrophobic interactions (Bukau and Horwich, 1998;Chen and Sigler, 1999;Shtilerman et al., 19...