Low Volume, microbatch-under oil style crystallization experiment

Nanovolume drops of protein solution are sufficient to fill an entire CrystalCard with ca. 800 individual crystallization experiments. Each aqueous droplet (plug) forms at the mixer where protein, buffer and a precipitant combine. The plugs spontaneously form when coming in contact with the inert, immiscible carrier fluid. : Plug Generation and Crystal formation

NO CROSS CONTAMINATION! A thin layer of carrier fluid between the plug and the wall of the microcapillary prevents any cross-contamination, making each plug a separate and distinct experiment.

  Diffraction-Ready^TM Crystals
 

Crystals that are grown within Peel-Apart™CrystalCards are directly accessible to manual harvesting.  A plastic seal is peeled off to give access to the crystals for extraction using a traditional cryo loop.  Alternatively, crystals may be subjected in-situ to X-ray diffraction. : Crystal Harvesting

  Optimization Screening

The MPCS™ uses on-chip formulation to generate fine concentration gradients of a crystallization condition.

Crystal growth optimization experiment with ribose-phosphate pyrophosphokinase Reference: Gerdts et. al. Acta Cryst. (2008). D64, 1116-1122

Very fine gradients are generated over a series of plugs to carefully interrogate crystallization phase space.

  Sparse Matrix Screening

Using the Hybrid Mode, a fine chemical gradient screen experiment is carried out on a variety of crystallization cocktails. This is a combination of a crystal hit via sparse matrix screening and subsequent optimization.

Pre-formed cartridges of precipitants seperated by gas bubbles are generated. The MPCS™ dispenses the pre-formed cartridges with a stream of protein solution and buffer to form a concentration gradient for each precipitant.

 In-situ X-ray Diffraction

 

Crystals grown in the CrystalCard were subjected in-situ to X-rays. Data collected at NE-CAT beamline 24ID-C at the Advanced Photon Source at Argonne National Laboratories. Data was merged from three different crystals, yielding an electron density map with a resolution of 1.9Å.

 
  Microfluidic Seeding

Microcrystals in volumes as little as 0.5 uL from a previous crystallization experiment can be used to carry out microfludic seeding in the MPCS™.

Microcrystals are aspirated into a Teflon tube (a). The microcrystal-containing solution is used as one of the aqueous streams in the 3+1 mixer (b). The CrystalCard™ is filled with plugs that each contain one or a few small microcrystals to seed crystal growth (c).

  No Dead Volume

Protein samples are used in their entirety in the protein crystallization trial. Prior to loading the protein solution, the syringe and Teflon tubing are back-filled with a carrier fluid (cf).
 

This back-filling method ensures that every nanoliter of protein sample is pumped into the CrystalCard.

 
  Recent Successes

The MPCS™was used at deCODE biostructures, Inc. to optimize the crystallization of an infectious disease target protein (Methionine-R-sulfoxide Reductase of Burkholderia pseudomallei).
Experiments were carried out at the Accelerated Technologies Center for Gene to 3D Structure in collaboration with the Seattle Structural Genomics Center for Infectious Diseases.

Crystals of methionine-R-sulfoxide reductase and methylisocitrate lyase were grown by performing gradient optimization experiments in MPCS™ CrystalCards™.  The efficiency of the structure determination process was improved while not requiring additional protein production.   Reference: Gerdts et. al. Acta Cryst. (2008). D64, 1116-1122 (1.7 Angstrom Structure deposited in PDB under 3CXK)