Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000–10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein-coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 [Formula: see text]g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs.

Introduction of a biotinylated monophosphine palladium complex within streptavidin affords an enantioselective artificial Suzukiase. Site-directed mutagenesis allowed the optimization of the activity and the enantioselectivity of this artificial metalloenzyme. A variety of atropisomeric biaryls were produced in good yields and up to 90% ee.

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X reveals 3D structures and elucidates functions of biomolecules with atomic resolution, thereby enabling researchers to make fundamental contributions to molecular biology and structure-based drug discovery 1. Synchrotron radiation, together with large-format 2D detectors, has been essential to the success of modern MX 2,3. In parallel with the evolution of synchrotron sources, several generations of X-ray detectors have been developed, namely, image plates 4 , multiwire proportional counters 5 , X-ray television detectors 6 , charge-coupled device (CCD) detectors 7 , and hybrid (pixel-array) photon-counting (HPC) detectors 8. Currently, most MX beamlines are equipped with HPC detectors, or are scheduled to be.

The molecular determinants responsible for the potency of the RXP470.1 phosphinic peptide inhibitor toward matrix metalloprotease-12 (MMP-12) remain elusive. To address this issue, structure-activity study, X-ray crystallography, and isothermal titration calorimetry (ITC) experiments were performed. The crystal structure of MMP-12/inhibitor complex (1.15 Å) reveals that the inhibitor establishes multiple interactions with the MMP-12 active site, with its long P(1)' side chain filling most of the S(1)' deep cavity. ITC experiments indicate that the binding of this inhibitor to MMP-12 is mostly entropy driven (ΔG° = -13.1 kcal/mol, ΔH° = -2.53 kcal/mol, and -TΔS° = -10.60 kcal/mol) and involves a proton uptake from the buffer. Comparing phosphinic versus hydroxamate inhibitors reveals that the chelation of the zinc ion is slightly different, leading the inhibitor backbone to adopt a position in which the hydrogen bonding with the MMP-12 active site is less favorable in phosphinic inhibitor while maintaining high affinity.

Molecular replacement in X-ray crystallography is the prime method for establishing structure-activity relationships of pharmaceutically relevant molecules. Such an approach is not available for NMR. Here, we establish a comparable method, called NMR molecular replacement (NMR(2)). The method requires experimentally measured ligand intramolecular NOEs and ligand-protein intermolecular NOEs as well as a previously known receptor structure or model. Our findings demonstrate that NMR(2) may open a new avenue for the fast and robust determination of the interaction site of ligand-protein complexes at atomic resolution.

Cryoprotection is the final step before flash-cooling, during which crystals can be improved or damaged and data quality maximized. A well thoughtout cryoprotection requires optimized composition of suitable components and an appropriate soak period. Crystallization methodology has been extensively studied, but not cryoprotection. Cryoprotectant selection remains a trial and error exercise where the first combination that "works" is accepted. The approach presented here consists in a formulation of a few mixed compounds to speed-up crystal preparation for synchrotron data collection. A good cryoprotectant solution needs to stabilize the crystalline state and prevent ice formation during flash-cooling, its composition can differ widely from the crystallization precipitant since it is not required to induce nucleation, establish an equilibrium between the protein crystal and the solution or permit crystal growth. The strategy presented here differs from the general practice that involves a single anti-icing chemical, often glycerol, a molecule able to enhance protein solubility. The multicomponent mixtures selected through an evolutive process neither increase nor decrease protein solubility but provide extended stabilization during cryoprotection to allow longer soak periods without crystal cracking or dissolving.