Single-particle cryo-electron microscopy of macromolecular assemblies

Sammanfattning: In this thesis, single-particle cryo-electron microscopy (cryo-EM) was used to study the structure of three macromolecular assemblies: the two hemocyanin isoforms from Rapana thomasiana, the Pyrococcus furiosus chaperonin, and the ribosome from Escherichia coli. Hemocyanins are large respiratory proteins in arthropods and molluscs. Most molluscan hemocyanins exist as two distinct isoforms composed of related polypeptides. In most species the two isoforms differ in terms of their oligomeric stability, and thus we set out to investigate the two Rapana thomasiana hemocyanins (RtH) in order to explain this behaviour. Our findings showed that the two RtHio forms are identical at the experimental resolution. Furthermore, three previously unreported connections that most likely contribute to the oligomeric stability were identified. Chaperonins are double-ring protein complexes that assist the folding process of nascent, non-native polypeptide chains. The chaperonin from the hyperthermophilic archaea Pyrococcus furiosus belongs to Group II chaperonins, and unlike most othergroup II chaperonins it appears to be homo-oligomeric. The 3D reconstruction of the Pyrococcus furiousus chaperonin revealed a di-octameric structure in a partially closed/open state, something in between the closed folding-active state and the open substrate-accepting state. The ribosome is the molecular machine where protein synthesis takes place. In bacteria there is a unique RNA molecule called transfer-messenger RNA (tmRNA) that together with its helper protein SmpB rescues ribosomes trapped on defective messenger RNAs (mRNAs) through a process called trans-translation. tmRNA is about 4 times the size of a normal tRNA, and it is composed of a tRNA-like domain (TLD) that is connected to the mRNA-like domain (MLD) by several pseudoknots (PKs) and RNA helices. During trans-translation, tmRNA utilize its TLD to receive the incomplete polypeptide from the peptidyl-tRNA in the ribosomal P site of the stalledribosome. Subsequently, its MLD is used to tag the incomplete polypeptide with adegradation signal. When tmRNA enters a stalled ribosome the MLD and pseudoknots form a highly structured arc that encircles the beak of the small ribosomal subunit. Byutilizing maximum-likelihood based methods for heterogeneity analysis we could observe the Escherichia coli ribosome in a number of different tmRNA·SmpB-boundstates. The cryo-EM map of the post-accommodated state revealed that the TLD·SmpBpart of the tmRNA·SmpB complex mimics native tRNAs in the A site of stalled ribosomes. The density map also showed that the tmRNA arc remains well structuredand that it is still attached to the beak of the small ribosomal subunit. Thereconstructions of the double-translocation tmRNA-bound ribosome complex showed that the pseudoknots of tmRNA still form an arc, and that they are located at positions similar to the ones assigned for the pseudoknots in the post-accommodated state. In addition, the tmRNA arc exists in two states; one stable and highly structured and another more flexible and disorganized.