3II9: Crystal Structure Of Glutaryl-Coa Dehydrogenase From Burkholderia Pseudomallei At 1.73 Angstrom

Citation:
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.
PDB ID: 3II9Download
MMDB ID: 78755
PDB Deposition Date: 2009/7/31
Updated in MMDB: 2009/12
Experimental Method:
x-ray diffraction
Resolution: 1.74  Å
Source Organism:
Similar Structures:
Biological Unit for 3II9: tetrameric; determined by author and by software (PISA)
Molecular Components in 3II9
Label Count Molecule
Proteins (4 molecules)
4
Glutaryl-coa Dehydrogenase
Molecule annotation
Chemicals (18 molecules)
1
4
2
4
3
2
4
3
5
5
* Click molecule labels to explore molecular sequence information.

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