Search PubMed⌕ Search
Home/Archive/PubMed

PubMed · 15039558

Assessing crystallization droplets using birefringence.

A Echalier·R L Glazer·V Fülöp·M A Geday
View original source ↗Export metadata ↓

Abstract

In this paper, the detection of crystalline elements in protein crystallization droplets containing precipitate is illustrated using the rotating-polarizer microscope technique. The sensitivity of this automated birefringence technique enables the detection of microcrystals in a precipitate that appears to be amorphous using traditional methods of inspection. The technique is illustrated with lysozyme and glucose isomerase. Glucose isomerase microcrystals were used successfully for seeding experiments and the conditions of both of the systems were refined to produce crystals suitable for X-ray analysis. The results are relevant to the field of high-throughput crystallography as an automated crystal-detection method as well as being a useful tool for detailed precipitate analysis.

Explore related subjects

Aldose-Ketose IsomerasesBirefringenceCrystallizationCrystallography, X-RayMuramidaseProteinsJournal ArticleResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

A Echalier, R L Glazer, V Fülöp, M A Geday. 2004-03-23. Assessing crystallization droplets using birefringence.. https://doi.org/10.1107/s0907444904003154

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2005

Isoprenoid biosynthesis as a target for antibacterial and antiparasitic drugs: phosphonohydroxamic acids as inhibitors of deoxyxylulose phosphate reducto-isomerase.

Isoprenoid biosynthesis via the methylerythritol phosphate pathway is a target against pathogenic bacteria and the malaria parasite Plasmodium falciparum. 4-(Hydroxyamino)-4-oxobutylphosphonic acid and 4-[hydroxy(methyl)amino]-4-oxobutyl phosphonic acid, two novel inhibitors of DXR (1-deoxy-D-xylulose 5-phosphate reducto-isomerase), the second enzyme of the pathway, have been synthesized and compared with fosmidomycin, the best known inhibitor of this enzyme. The latter phosphonohydroxamic acid showed a high inhibitory activity towards DXR, much like fosmidomycin, as well as significant antibacterial activity against Escherichia coli in tests on Petri dishes.

Aldose-Ketose Isomerases↗
PubMed2004

Substrate analogs for the investigation of deoxyxylulose 5-phosphate reductoisomerase inhibition: synthesis and evaluation.

Deoxyxylulose 5-phosphate (DXP) analogs were synthesized and evaluated as alternative substrates and inhibitors of recombinant Synechocystis PCC6803 DXP reductoisomerase (DXR; EC 1.1.1.267). Five of the compounds tested (1,2-dideoxy-D-threo-3-hexulose 6-phosphate, 1-deoxy-l-ribulose 5-phosphate, 2S,3R-dihydroxybutyramide 4-phosphate, 4S-hydroxypentan-2-one 5-phosphate, and 3S-hydroxypentan-2-one 5-phosphate) acted as relatively weak competitive inhibitors when compared to fosmidomycin. A sixth compound, 3R,4S-dihydroxy-5-oxohexylphosphonic acid, served as an alternate substrate, as has recently been reported for the same compound with Escherichia coli DXR.

Aldose-Ketose Isomerases↗
PubMed2004

Structure of 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase from Shewanella oneidensis at 1.6 A: identification of farnesyl pyrophosphate trapped in a hydrophobic cavity.

Isopentenyl pyrophosphate (IPP) is a universal building block for the ubiquitous isoprenoids that are essential to all organisms. The enzymes of the non-mevalonate pathway for IPP synthesis, which is unique to many pathogenic bacteria, have recently been explored as targets for antibiotic development. Several crystal structures of 2C-methyl-D-erythritol-2,4-cyclophosphate (MECDP) synthase, the fifth of seven enzymes involved in the non-mevalonate pathway for synthesis of IPP, have been reported; however, the composition of metal ions in the active site and the presence of a hydrophobic cavity along the non-crystallographic threefold symmetry axis has varied between the reported structures. Here, the structure of MEDCP from Shewanella oneidensis MR1 (SO3437) was determined to 1.6 A resolution in the absence of substrate. The presence of a zinc ion in the active-site cleft, tetrahedrally coordinated by two histidine side chains, an aspartic acid side chain and an ambiguous fourth ligand, was confirmed by zinc anomalous diffraction. Based on analysis of anomalous diffraction data and typical metal-to-ligand bond lengths, it was concluded that an octahedral sodium ion was 3.94 A from the zinc ion. A hydrophobic cavity was observed along the threefold non-crystallographic symmetry axis, filled by a well defined non-protein electron density that could be modeled as farnesyl pyrophosphate (FPP), a downstream product of IPP, suggesting a possible feedback mechanism for enzyme regulation. The high-resolution data clarified the FPP-binding mode compared with previously reported structures. Multiple sequence alignment indicated that the residues critical to the formation of the hydrophobic cavity and for coordinating the pyrophosphate group of FPP are present in the majority of MEDCP synthase enzymes, supporting the idea of a specialized biological function related to FPP binding in a subfamily of MEDCP synthase homologs.

Aldose-Ketose Isomerases↗