Oxygen-Sensitive Metalloprotein Structure Determination by Cryo-Electron Microscopy.

Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions.

Methods to Screen for Radical SAM Enzyme Crystallization Conditions.

Radical S-adenosyl-L-methionine proteins most probably belong to the widest superfamily of metalloenzymes. Thanks to their ability to catalyze difficult reactions, combined with their involvement in the biosynthesis of numbers of natural products, they sound promising for various biotechnological applications. Their structural study is often limited because they are usually challenging to crystallize.

Structural Insights into the Mechanism of the Radical SAM Carbide Synthase NifB, a Key Nitrogenase Cofactor Maturating Enzyme.

Nitrogenase is a key player in the global nitrogen cycle, as it catalyzes the reduction of dinitrogen into ammonia. The active site of the nitrogenase MoFe protein corresponds to a [MoFeSC-()-homocitrate] species designated FeMo-cofactor, whose biosynthesis and insertion requires the action of over a dozen maturation proteins provided by the NIF (for trogen ixation) assembly machinery. Among them, the radical SAM protein NifB plays an essential role, concomitantly inserting a carbide ion and coupling two [FeS] clusters to form a [FeSC] precursor called NifB-co.

REACH: Robotic Equipment for Automated Crystal Harvesting using a six-axis robot arm and a micro-gripper.

In protein crystallography experiments, only two critical steps remain manual: the transfer of crystals from their original crystallization drop into the cryoprotection solution followed by flash-cooling. These steps are risky and tedious, requiring a high degree of manual dexterity. These limiting steps are a real bottleneck to high-throughput crystallography and limit the remote use of protein crystallography core facilities.

Simultaneous measurements of solvent dynamics and functional kinetics in a light-activated enzyme.

Solvent fluctuations play a key role in controlling protein motions and biological function. Here, we have studied how individual steps of the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (POR) couple with solvent dynamics. To simultaneously monitor the catalytic cycle of the enzyme and the dynamical behavior of the solvent, we designed temperature-dependent UV-visible microspectrophotometry experiments, using flash-cooled nanodroplets of POR to which an exogenous soluble fluorophore was added.