5D57: In Meso X-ray Crystallography Structure Of Diacylglycerol Kinase, Dgka, At 100 K

Citation:
Abstract
Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, alpha-helical and beta-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins.
PDB ID: 5D57Download
MMDB ID: 135465
PDB Deposition Date: 2015/8/10
Updated in MMDB: 2016/03
Experimental Method:
x-ray diffraction
Resolution: 2.8  Å
Source Organism:
Similar Structures:
Biological Unit for 5D57: trimeric; determined by author and by software (PISA)
Molecular Components in 5D57
Label Count Molecule
Proteins (3 molecules)
3
Diacylglycerol Kinase(Gene symbol: dgkA)
Molecule annotation
Chemicals (14 molecules)
1
14
* Click molecule labels to explore molecular sequence information.

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