Abstract:The MPCS Plug Maker is a microcapillary‐based protein‐crystallization system for generating diffraction‐ready crystals from nanovolumes of protein. Crystallization screening using the Plug Maker was used as a salvage pathway for proteins that failed to crystallize during the initial observation period using the traditional sitting‐drop vapor‐diffusion method. Furthermore, the CrystalCards used to store the crystallization experiments set up by the Plug Maker are shown be a viable container for long‐term storag… Show more
“…For in situ data collection, the capillary containing the droplets was cut and sealed, and fixed on a magnetic base. The commercial CrystalCard device, 75,76 by Protein BioSolutions, works on the same principle. The crystals produced can be harvested or measured in situ, either directly inside the chip or by coupling with a capillary.…”
Section: Microfluidic Methods For In Situmentioning
“…For in situ data collection, the capillary containing the droplets was cut and sealed, and fixed on a magnetic base. The commercial CrystalCard device, 75,76 by Protein BioSolutions, works on the same principle. The crystals produced can be harvested or measured in situ, either directly inside the chip or by coupling with a capillary.…”
Section: Microfluidic Methods For In Situmentioning
“…0.4 ml protein solution was mixed with 0.4 ml reservoir solution and equilibrated against 100 ml reservoir solution using 96-well Compact Jr plates from Emerald BioSystems. The protein solution consisted of 11 mg ml À1 BbDHFR-TS in SEC buffer and crystal conditions were optimized using the Microcapillary Protein Crystallization System (MPCS) from Emerald BioSystems (Gerdts et al, 2008(Gerdts et al, , 2010Christensen et al, 2011). The BbDHFR-TS crystal used to solve the apo structure (PDB entry 3i3r) was obtained from a crystal card running a microfluidic gradient focused at 20%(w/v) polyethylene glycol (PEG) 8000 and 100 mM N-cyclohexyl-2-aminoethanesulfonic acid (CHES) pH 9.5.…”
Babesiosis is a tick‐borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase‐thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase‐thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug‐resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research.
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