Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody

Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein

The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry

AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ∼20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH

New insights on the structure of alpha-synuclein fibrils using cryo-electron microscopy

Fibrils of alpha-synuclein are significant components of cellular inclusions associated with several neuropathological disorders including Parkinson’s disease, multiple system atrophy and dementia with Lewy bodies. In recent years, technological advances in the field of transmission electron microscopy and image processing have made it possible to solve the structure of alpha-synuclein fibrils at high resolution. This review discusses the results of structural studies using cryo-electron microscopy, which revealed that in-vitro produced fibrils vary in diameter from 5 nm for single-protofilament fibrils, to 10 nm for two-protofilament fibrils. In addition, the atomic models hint at contributions of the familial Parkinson’s disease mutation sites to inter-protofilament interaction and the locations where post-translational modifications take place. Here, we propose a nomenclature system that allows identifying the existing alpha-synuclein polymorphs and that will allow to incorporate additional high-resolution structures determined in the future

CHARACTERIZATION OF TEN STRAINS OF FILAMENTOUS CYANOBACTERIA FROM THE SOUTH SHETLAND ISLANDS, MARITIME ANTARCTICA

The evolutionary relationships of ten Antarctic cyanobacterial strains of the order Oscillatoriales isolated from King George Island and Deception Island, South Shetland Islands were studied by a polyphasic approach. Phenotypic observations of the morphological features and genotypic analyses (16S rRNA and ITS sequences) were performed. Based on major phenotypic features, the strains were divided into four distinct morphotypes: Leptolyngbya borchgrevinkii (A), Leptolyngbya frigida (B), Phormidium autumnale (C) and Wilmottia murrayi (D). This morphological identification was in global agreement with the evolutionary relationships. According to the phylogenetic analysis, the ten strains were divided into two major clades, containing related strain sequences with Leptolyngbya morphotypes in one clade and with morphotypes corresponding to Phormidium, Wilmottia and Microcoleus spp. in the other clade. Each major clade was divided into two sub-clades. For the first time, the 16S rRNA gene sequence of a strain corresponding to the Leptolyngbya borchgrevinkii morphotype (A) was determined, on the basis of strain KOVACIK-ANT 1990/4. The closest sequence to our morphotype A is the clone Fr252 isolated from microbial mat of Antarctic Lake Fryxell. Morphotype B is closest to sequences assigned to Leptolyngbya frigida isolated from microbial mats of lakes in continental East Antarctica. Morphotype C belongs to a cluster including strains with morphotypes corresponding to Phormidium autumnale from Antarctica, but also from Europe. Morphotype D is grouped with sequences of the morphotype assigned to Wilmottia murrayi isolated from Antarctica.CCAMBIO, BIPOLES

Cryo-EM structure of phosphodiesterase 6 reveals insights into the allosteric regulation of type I phosphodiesterases

Cyclic nucleotide phosphodiesterases (PDEs) work in conjunction with adenylate/guanylate cyclases to regulate the key second messengers of G protein-coupled receptor signaling. Previous attempts to determine the full-length structure of PDE family members at high-resolution have been hindered by structural flexibility, especially in their linker regions and N- and C-terminal ends. Therefore, most structure-activity relationship studies have so far focused on truncated and conserved catalytic domains rather than the regulatory domains that allosterically govern the activity of most PDEs. Here, we used single-particle cryo-electron microscopy to determine the structure of the full-length PDE6αβ2γ complex. The final density map resolved at 3.4 Å reveals several previously unseen structural features, including a coiled N-terminal domain and the interface of PDE6γ subunits with the PDE6αβ heterodimer. Comparison of the PDE6αβ2γ complex with the closed state of PDE2A sheds light on the conformational changes associated with the allosteric activation of type I PDEs

Polyphasic characterization of 10 selected ecologically relevant filamentous cyanobacterial strains from the South Shetland Islands, Maritime Antarctica

The evolutionary relationships of 10 Antarctic cyanobacterial strains of the order Oscillatoriales isolated from King George and Deception Islands, South Shetland Islands were studied by a polyphasic approach (morphology, 16S rRNA and internal transcribed spacer sequences). The studied taxa are characteristic of coastal Antarctic biotopes, where they form distinct populations and ecologically delimited communities. They were isolated from terrestrial habitats: microbial mats in seepages; crusts on soil, rocks, bones and mosses; mud, sometimes close to bird colonies; and from guano. Based on major phenotypic features, the strains were divided into four distinct morphotypes: Leptolyngbya borchgrevinkii (A), Leptolyngbya frigida (B), Microcoleus sp. (C) and Wilmottia murrayi (D). This morphological identification was in agreement with the phylogenetic relationships. For the first time, the 16S rRNA gene sequence of a strain corresponding to the L. borchgrevinkii morphotype was determined. Morphotype B is most related to sequences assigned to L. frigida isolated from microbial mats of coastal lakes in East Antarctica. Morphotype C belongs to a cluster including strains with morphotypes corresponding to Microcoleus attenuatus, Microcoleus favosus and Microcoleus sp., which are from Antarctica and other continents. Morphotype D is grouped with sequences assigned to W. murrayi mostly isolated from Antarctica.Biodiversity of Antarctic cyanobacteri

Microfluidic protein isolation and sample preparation for high-resolution cryo-EM

High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 mu L of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes