In situ high-resolution structure of the baseplate antenna complex in Chlorobaculum tepidum.

Photosynthetic antenna systems enable organisms harvesting light and transfer the energy to the photosynthetic reaction centre, where the conversion to chemical energy takes place. One of the most complex antenna systems, the chlorosome, found in the photosynthetic green sulfur bacterium Chlorobaculum (Cba.) tepidum contains a baseplate, which is a scaffolding super-structure, formed by the protein CsmA and bacteriochlorophyll a. Here we present the first high-resolution structure of the CsmA baseplate using intact fully functional, light-harvesting organelles from Cba. tepidum, following a hybrid approach combining five complementary methods: solid-state NMR spectroscopy, cryo-electron microscopy, isotropic and anisotropic circular dichroism and linear dichroism. The structure calculation was facilitated through development of new software, GASyCS for efficient geometry optimization of highly symmetric oligomeric structures. We show that the baseplate is composed of rods of repeated dimers of the strongly amphipathic CsmA with pigments sandwiched within the dimer at the hydrophobic side of the helix.

tmRNA to the rescue: structural motives for the salvage of stalled ribosomes.

During translation, mRNA molecules are incidentally damaged, leaving the ribosome unable to reach or recognize the stop codon and thus stalled with mRNA and a potentially harmful polypeptide product attached to tRNA in the ribosomal P-site. In bacteria, a process called trans-translation has evolved, where a protein-RNA complex (smpB-tmRNA) mimicks the role of aminoacyl charged tRNA, replacing stalled tRNA in the ribosomal A-site. The ribosome then resumes protein synthesis guided by an mRNA-like portion of the tmRNA which ends with a stop codon, and codes for a peptide sequence susceptible to proteolysis, thus allowing the bacteria to salvage stalled ribosomes and degrade ill-defined and potentially harmful protein products. In this article, we will recollect how structural studies have yielded a model for how the pre-translocation stages of trans-translation employing structural mimicry. We will also discuss possible models for how the translocation may be carried out.

ATP-induced conformational dynamics in the AAA+ motor unit of magnesium chelatase.

Mg-chelatase catalyzes the first committed step of the chlorophyll biosynthetic pathway, the ATP-dependent insertion of Mg(2+) into protoporphyrin IX (PPIX). Here we report the reconstruction using single-particle cryo-electron microscopy of the complex between subunits BchD and BchI of Rhodobacter capsulatus Mg-chelatase in the presence of ADP, the nonhydrolyzable ATP analog AMPPNP, and ATP at 7.5 A, 14 A, and 13 A resolution, respectively. We show that the two AAA+ modules of the subunits form a unique complex of 3 dimers related by a three-fold axis. The reconstructions demonstrate substantial differences between the conformations of the complex in the presence of ATP and ADP, and suggest that the C-terminal integrin-I domains of the BchD subunits play a central role in transmitting conformational changes of BchI to BchD. Based on these data a model for the function of magnesium chelatase is proposed.

tmRNA.SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes.

Bacterial ribosomes stalled on faulty, often truncated, mRNAs lacking stop codons are rescued by trans-translation. It relies on an RNA molecule (tmRNA) capable of replacing the faulty mRNA with its own open reading frame (ORF). Translation of tmRNA ORF results in the tagging of faulty protein for degradation and its release from the ribosome. We used single-particle cryo-electron microscopy to visualize tmRNA together with its helper protein SmpB on the 70S Escherichia coli ribosome in states subsequent to GTP hydrolysis on elongation factor Tu (EF-Tu). Three-dimensional reconstruction and heterogeneity analysis resulted in a 15A resolution structure of the tmRNA.SmpB complex accommodated in the A site of the ribosome, which shows that SmpB mimics the anticodon- and D-stem of native tRNAs missing in the tRNA-like domain of tmRNA. We conclude that the tmRNA.SmpB complex accommodates in the ribosomal A site very much like an aminoacyl-tRNA during protein elongation.

Cryo-EM reveals promoter DNA binding and conformational flexibility of the general transcription factor TFIID.

The general transcription factor IID (TFIID) is required for initiation of RNA polymerase II-dependent transcription at many eukaryotic promoters. TFIID comprises the TATA-binding protein (TBP) and several conserved TBP-associated factors (TAFs). Recognition of the core promoter by TFIID assists assembly of the preinitiation complex. Using cryo-electron microscopy in combination with methods for ab initio single-particle reconstruction and heterogeneity analysis, we have produced density maps of two conformational states of Schizosaccharomyces pombe TFIID, containing and lacking TBP. We report that TBP-binding is coupled to a massive histone-fold domain rearrangement. Moreover, docking of the TBP-TAF1(N-terminus) atomic structure to the TFIID map and reconstruction of a TAF-promoter DNA complex helps to account for TAF-dependent regulation of promoter-TBP and promoter-TAF interactions.

Structural basis of the iron storage function of frataxin from single-particle reconstruction of the iron-loaded oligomer.

The mitochondrial protein frataxin plays a central role in mitochondrial iron homeostasis, and frataxin deficiency is responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1 in 40000 children. Here we present a single-particle reconstruction from cryoelectron microscopic images of iron-loaded 24-subunit oligomeric frataxin particles at 13 and 17 A resolution. Computer-aided classification of particle images showed heterogeneity in particle size, which was hypothesized to result from gradual accumulation of iron within the core structure. Thus, two reconstructions were created from two classes of particles with iron cores of different sizes. The reconstructions show the iron core of frataxin for the first time. Compared to the previous reconstruction of iron-free particles from negatively stained images, the higher resolution of the present reconstruction allowed a more reliable analysis of the overall three-dimensional structure of the 24-meric assembly. This was done after docking the X-ray structure of the frataxin trimer into the EM reconstruction. The structure revealed a close proximity of the suggested ferroxidation sites of different monomers to the site proposed to serve in iron nucleation and mineralization. The model also assigns a new role to the N-terminal helix of frataxin in controlling the channel at the 4-fold axis of the 24-subunit oligomer. The reconstructions show that, together with some common features, frataxin has several unique features which distinguish it from ferritin. These include the overall organization of the oligomers, the way they are stabilized, and the mechanisms of iron core nucleation.

A new cryo-EM single-particle ab initio reconstruction method visualizes secondary structure elements in an ATP-fueled AAA+ motor.

The generation of ab initio three-dimensional (3D) models is a bottleneck in the studies of large macromolecular assemblies by single-particle cryo-electron microscopy. We describe here a novel method, in which established methods for two-dimensional image processing are combined with newly developed programs for joint rotational 3D alignment of a large number of class averages (RAD) and calculation of 3D volumes from aligned projections (VolRec). We demonstrate the power of the method by reconstructing an approximately 660-kDa ATP-fueled AAA+ motor to 7.5 A resolution, with secondary structure elements identified throughout the structure. We propose the method as a generally applicable automated strategy to obtain 3D reconstructions from unstained single particles imaged in vitreous ice.

The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II.

CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II.

The structures of frataxin oligomers reveal the mechanism for the delivery and detoxification of iron.

Defects in the mitochondrial protein frataxin are responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1:40,000 children. Here, we present the crystal structures of the iron-free and iron-loaded frataxin trimers, and a single-particle electron microscopy reconstruction of a 24 subunit oligomer. The structures reveal fundamental aspects of the frataxin mechanism. The trimer has a central channel in which one atom of iron binds. Two conformations of the channel with different metal-binding affinities suggest that a gating mechanism controls whether the bound iron is delivered to other proteins or transferred to detoxification sites. The trimer constitutes the basic structural unit of the 24 subunit oligomer. The architecture of this oligomer and several features of the trimer structure demonstrate striking similarities to the iron-storage protein ferritin. The data reveal how stepwise assembly provides frataxin with the structural flexibility to perform two functions: metal delivery and detoxification.