3II9

Crystal structure of glutaryl-coa dehydrogenase from Burkholderia pseudomallei at 1.73 Angstrom

Summary for 3II9

• Entry DOI: 10.2210/pdb3ii9/pdb
• Related: 3D6B 3EOM
• Descriptor: Glutaryl-CoA dehydrogenase, DI(HYDROXYETHYL)ETHER, MAGNESIUM ION, ... (7 entities in total)
• Functional Keywords: slipchip, microfluidics, screening, optimization, protein crystallization, structural genomics, psi-2, protein structure initiative, accelerated technologies center for gene to 3d structure, atcg3d, fad, flavoprotein, oxidoreductase, seattle structural genomics center for infectious disease, ssgcid
• Biological source: Burkholderia pseudomallei 1710b
• Total number of polymer chains: 4
• Total formula weight: 174890.51

Authors

Ismagilov, R.F.,Li, L.,Du, W.B.,Staker, B.,Accelerated Technologies Center for Gene to 3D Structure (ATCG3D),Seattle Structural Genomics Center for Infectious Disease (SSGCID) (deposition date: 2009-07-31, release date: 2009-12-15, Last modification date: 2023-09-06)

Primary citation

Li, L.,Du, W.,Ismagilov, R.
User-loaded SlipChip for equipment-free multiplexed nanoliter-scale experiments.
J.Am.Chem.Soc., 132:106-111, 2010

PubMed Abstract: This paper describes a microfluidic approach to perform multiplexed nanoliter-scale experiments by combining a sample with multiple different reagents, each at multiple mixing ratios. This approach employs a user-loaded, equipment-free SlipChip. The mixing ratios, characterized by diluting a fluorescent dye, could be controlled by the volume of each of the combined wells. The SlipChip design was validated on an approximately 12 nL scale by screening the conditions for crystallization of glutaryl-CoA dehydrogenase from Burkholderia pseudomallei against 48 different reagents; each reagent was tested at 11 different mixing ratios, for a total of 528 crystallization trials. The total consumption of the protein sample was approximately 10 microL. Conditions for crystallization were successfully identified. The crystallization experiments were successfully scaled up in well plates using the conditions identified in the SlipChip. Crystals were characterized by X-ray diffraction and provided a protein structure in a different space group and at a higher resolution than the structure obtained by conventional methods. In this work, this user-loaded SlipChip has been shown to reliably handle fluids of diverse physicochemical properties, such as viscosities and surface tensions. Quantitative measurements of fluorescent intensities and high-resolution imaging were straighforward to perform in these glass SlipChips. Surface chemistry was controlled using fluorinated lubricating fluid, analogous to the fluorinated carrier fluid used in plug-based crystallization. Thus, we expect this approach to be valuable in a number of areas beyond protein crystallization, especially those areas where droplet-based microfluidic systems have demonstrated successes, including measurements of enzyme kinetics and blood coagulation, cell-based assays, and chemical reactions.

PubMed: 20000708
DOI: 10.1021/ja908555n

Experimental method

X-RAY DIFFRACTION (1.74 Å)