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Large assemblies During recent years a significant progress with regard to resolution in cryoEM studies of single particles has been achieved. Virus structures are approaching resolutions which will allow tracing of polypeptide chains of capsid proteins. Completely asymmetric assemblies like ribosomes are now also studied at this level. A particularly interesting approach for large complexes is to study individual components by X-ray crystallography and/or NMR and dock the structures onto cryoEM maps of the complete assemblies. The specific research program for large assemblies is at present focused on one chaperone and a hemocyanin complex: Pyrrococcus furiosus chaperone In colaboration with the in-house X-ray group of Rudolf Ladenstein we study the chaperone of the hyperthermophilic archeon Pyrrococcus furiosus. This protein complex of 1 MDa mass is beleived to be a homo-di-octamer. We have purified the protein from an E.coli expression system sufficiently for electron microscopy and cryo-EM studies are underway. Our preliminary negative stain reconstruction confirms the proposed D8-symmetry and the overall architecture of archeal chaperones. An interesting difference to the well-studied thermosome is that fact that this oligomer consists of only one type of subunit. This might have evolutionary implications and at a very practical level, due to the higher symmetry makes it more suitable for high-resolution studies.   Fig 1: Side and top-view of the chaperone from Pyrrococcus furiosus Rapana thomasiana hemocyanins In collaboration with the Institute of Organic Chemistry of the Bulgarian Academy of Sciences in Sofia we study the 8 MDa respiratory protein of the gastropod Rapana thomasiana. This protein exists as two structurally distinct homo-didecamers, hemocyanin 1 and 2. The group in Sofia has purified each of the two forms seperately as well as the native mixture from the organism. A preliminary negative stain study at 30Å resolution confirms the D5 symmetry also found in the same molecule from other organisms. Clear similarities betwee H1 and H2 have been found (the arc-structure) as well clear differences especially in the ”collar structure”. The native mixture of H1 and H2 does indeed appear as an average of the H1 and the H2 structure.
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