Towards a molecular picture of the archaeal cell surface.

Archaea produce various protein filaments with specialised functions. While some archaea produce only one type of filament, the archaeal model species Sulfolobus acidocaldarius generates four. These include rotary swimming propellers analogous to bacterial flagella (archaella), pili for twitching motility (Aap), adhesive fibres (threads), and filaments facilitating homologous recombination upon UV stress (UV pili).

CryoEM reveals the structure of an archaeal pilus involved in twitching motility.

Amongst the major types of archaeal filaments, several have been shown to closely resemble bacterial homologues of the Type IV pili (T4P). Within Sulfolobales, member species encode for three types of T4P, namely the archaellum, the UV-inducible pilus system (Ups) and the archaeal adhesive pilus (Aap). Whereas the archaellum functions primarily in swimming motility, and the Ups in UV-induced cell aggregation and DNA-exchange, the Aap plays an important role in adhesion and twitching motility.

Structure of the two-component S-layer of the archaeon .

Surface layers (S-layers) are resilient two-dimensional protein lattices that encapsulate many bacteria and most archaea. In archaea, S-layers usually form the only structural component of the cell wall and thus act as the final frontier between the cell and its environment. Therefore, S-layers are crucial for supporting microbial life.

Structural characterization of a novel cyclic 2,3-diphosphoglycerate synthetase involved in extremolyte production in the archaeon .

The enzyme cyclic di-phosphoglycerate synthetase that is involved in the production of the osmolyte cyclic 2,3-diphosphoglycerate has been studied both biochemically and structurally. Cyclic 2,3-diphosphoglycerate is found exclusively in the hyperthermophilic archaeal methanogens, such as , , and . Its presence increases the thermostability of archaeal proteins and protects the DNA against oxidative damage caused by hydroxyl radicals.

CryoEM reveals that ribosomes in microsporidian spores are locked in a dimeric hibernating state.

Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of electron cryo-tomography and single-particle electron cryo-microscopy.

Structure-function analysis of ceTIR-1/hSARM1 explains the lack of Wallerian axonal degeneration in C. elegans.

Wallerian axonal degeneration (WD) does not occur in the nematode C. elegans, in contrast to other model animals. However, WD depends on the NADase activity of SARM1, a protein that is also expressed in C.

Structural insights into the DNA recognition mechanism by the bacterial transcription factor PdxR.

Specificity in protein-DNA recognition arises from the synergy of several factors that stem from the structural and chemical signatures encoded within the targeted DNA molecule. Here, we deciphered the nature of the interactions driving DNA recognition and binding by the bacterial transcription factor PdxR, a member of the MocR family responsible for the regulation of pyridoxal 5'-phosphate (PLP) biosynthesis. Single particle cryo-EM performed on the PLP-PdxR bound to its target DNA enabled the isolation of three conformers of the complex, which may be considered as snapshots of the binding process.

The ROK kinase N-acetylglucosamine kinase uses a sequential random enzyme mechanism with successive conformational changes upon each substrate binding.

N-acetyl-d-glucosamine (GlcNAc) is a major component of bacterial cell walls. Many organisms recycle GlcNAc from the cell wall or metabolize environmental GlcNAc. The first step in GlcNAc metabolism is phosphorylation to GlcNAc-6-phosphate.

Electron cryo-microscopy reveals the structure of the archaeal thread filament.

Pili are filamentous surface extensions that play roles in bacterial and archaeal cellular processes such as adhesion, biofilm formation, motility, cell-cell communication, DNA uptake and horizontal gene transfer. The model archaeaon Sulfolobus acidocaldarius assembles three filaments of the type-IV pilus superfamily (archaella, archaeal adhesion pili and UV-inducible pili), as well as a so-far uncharacterised fourth filament, named "thread". Here, we report on the cryo-EM structure of the archaeal thread.

CCP4 Cloud for structure determination and project management in macromolecular crystallography.

Nowadays, progress in the determination of three-dimensional macromolecular structures from diffraction images is achieved partly at the cost of increasing data volumes. This is due to the deployment of modern high-speed, high-resolution detectors, the increased complexity and variety of crystallographic software, the use of extensive databases and high-performance computing. This limits what can be accomplished with personal, offline, computing equipment in terms of both productivity and maintainability.

Escaping the symmetry trap in helical reconstruction.

Helical reconstruction is the method of choice for obtaining 3D structures of filaments from electron cryo-microscopy (cryoEM) projections. This approach relies on applying helical symmetry parameters deduced from Fourier-Bessel or real space analysis, such as sub-tomogram averaging. While helical reconstruction continues to provide invaluable structural insights into filaments, its inherent dependence on imposing a pre-defined helical symmetry can also introduce bias.

Spinning sugars in antigen biosynthesis: characterization of the Coxiella burnetii and Streptomyces griseus TDP-sugar epimerases.

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central moiety of the antibiotic streptomycin, while DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii.

An archaellum filament composed of two alternating subunits.

Archaea use a molecular machine, called the archaellum, to swim. The archaellum consists of an ATP-powered intracellular motor that drives the rotation of an extracellular filament composed of multiple copies of proteins named archaellins. In many species, several archaellin homologs are encoded in the same operon; however, previous structural studies indicated that archaellum filaments mainly consist of only one protein species.

Structural basis for UFM1 transfer from UBA5 to UFC1.

Ufmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear.

Biochemical and Structural Characterisation of a Novel D-Lyxose Isomerase From the Hyperthermophilic Archaeon sp.

A novel D-lyxose isomerase has been identified within the genome of a hyperthermophilic archaeon belonging to the species. The enzyme has been cloned and over-expressed in and biochemically characterised. This enzyme differs from other enzymes of this class in that it is highly specific for the substrate D-lyxose, showing less than 2% activity towards mannose and other substrates reported for lyxose isomerases.

Structural Insights into a Novel Esterase from the East Pacific Rise and Its Improved Thermostability by a Semirational Design.

Lipolytic enzymes are essential biocatalysts in food processing as well as pharmaceutical and pesticide industries, catalyzing the cleavage of ester bonds in a variety of acyl chain substrates. Here, we report the crystal structure of an esterase from the deep-sea hydrothermal vent of the East Pacific Rise (EprEst). The X-ray structure of EprEst in complex with the ligand, acetate, has been determined at 2.

A 'Split-Gene' Transketolase From the Hyper-Thermophilic Bacterium : Structure and Biochemical Characterization.

A novel transketolase has been reconstituted from two separate polypeptide chains encoded by a 'split-gene' identified in the genome of the hyperthermophilic bacterium, . The reconstituted active αβ tetrameric enzyme has been biochemically characterized and its activity has been determined using a range of aldehydes including glycolaldehyde, phenylacetaldehyde and cyclohexanecarboxaldehyde as the ketol acceptor and hydroxypyruvate as the donor. This reaction proceeds to near 100% completion due to the release of the product carbon dioxide and can be used for the synthesis of a range of sugars of interest to the pharmaceutical industry.

Structural basis for SARM1 inhibition and activation under energetic stress.

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.

Structural insights into the NAD-dependent formate dehydrogenase mechanism revealed from the NADH complex and the formate NAD ternary complex of the Chaetomium thermophilum enzyme.

The removal of carbon dioxide from the waste streams of industrial processes is a major challenge for creation of a sustainable circular economy. This makes the synthesis of formate from CO by NAD dependent formate dehydrogenases (FDHs) an attractive process for this purpose. The efficiency of this reaction is however low and to achieve a viable industrial process an optimised engineered enzyme needs to be developed.

The crystal structure of Arabidopsis BON1 provides insights into the copine protein family.

The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium-dependent membrane-binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure.

Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases.

Biocatalysis, the use of enzymes in chemical transformations, is an important green chemistry tool. Cascade reactions combine different enzyme activities in a sequential set of reactions. Cascades can occur within a living (usually bacterial) cell; in vitro in 'one pot' systems where the desired enzymes are mixed together to carry out the multi-enzyme reaction; or using microfluidic systems.

The crystal structure of human microsomal triglyceride transfer protein.

Microsomal triglyceride transfer protein (MTP) plays an essential role in lipid metabolism, especially in the biogenesis of very low-density lipoproteins and chylomicrons via the transfer of neutral lipids and the assembly of apoB-containing lipoproteins. Our understanding of the molecular mechanisms of MTP has been hindered by a lack of structural information of this heterodimeric complex comprising an MTPα subunit and a protein disulfide isomerase (PDI) β-subunit. The structure of MTP presented here gives important insights into the potential mechanisms of action of this essential lipid transfer molecule, structure-based rationale for previously reported disease-causing mutations, and a means for rational drug design against cardiovascular disease and obesity.

Structural Evidence for an Octameric Ring Arrangement of SARM1.

SARM1 induces axonal degeneration in response to various insults and is therefore considered an attractive drug target for the treatment of neuro-degenerative diseases as well as for brain and spinal cord injuries. SARM1 activity depends on the integrity of the protein's SAM domains, as well as on the enzymatic conversion of NAD+ to ADPR (ADP Ribose) products by the SARM1's TIR domain. Therefore, inhibition of either SAM or TIR functions may constitute an effective therapeutic strategy.