3 research outputs found
Protein Crystallization in an Actuated Microfluidic Nanowell Device
Protein crystallization is a major
bottleneck of structure determination
by X-ray crystallography, hampering the process by years in some cases.
Numerous matrix screening trials using significant amounts of protein
are often applied, while a systematic approach with phase diagram
determination is prohibited for many proteins that can only be expressed
in small amounts. Here, we demonstrate a microfluidic nanowell device
implementing protein crystallization and phase diagram screening using
nanoscale volumes of protein solution per trial. The device is made
with cost-effective materials and is completely automated for efficient
and economical experimentation. In the developed device, 170 trials
can be realized with unique concentrations of protein and precipitant
established by gradient generation and isolated by elastomeric valving
for crystallization incubation. Moreover, this device can be further
downscaled to smaller nanowell volumes and larger scale integration.
The device was calibrated using a fluorescent dye and compared to
a numerical model where concentrations of each trial can be quantified
to establish crystallization phase diagrams. Using this device, we
successfully crystallized lysozyme and C-phycocyanin, as visualized
by compatible crystal imaging techniques such as bright-field microscopy,
UV fluorescence, and second-order nonlinear imaging of chiral crystals.
Concentrations yielding observed crystal formation were quantified
and used to determine regions of the crystallization phase space for
both proteins. Low sample consumption and compatibility with a variety
of proteins and imaging techniques make this device a powerful tool
for systematic crystallization studies
High Throughput Protein Nanocrystal Fractionation in a Microfluidic Sorter
Protein
crystallography is transitioning into a new generation
with the introduction of the X-ray free electron laser, which can
be used to solve the structures of complex proteins via serial femtosecond
crystallography. Sample characteristics play a critical role in successful
implementation of this new technology, whereby a small, narrow protein
crystal size distribution is desired to provide high quality diffraction
data. To provide such a sample, we developed a microfluidic device
that facilitates dielectrophoretic sorting of heterogeneous particle
mixtures into various size fractions. The first generation device
demonstrated great potential and success toward this endeavor; thus,
in this work, we present a comprehensive optimization study to improve
throughput and control over sorting outcomes. First, device geometry
was designed considering a variety of criteria, and applied potentials
were modeled to determine the scheme achieving the largest sorting
efficiency for isolating nanoparticles from microparticles. Further,
to investigate sorting efficiency within the nanoparticle regime,
critical geometrical dimensions and input parameters were optimized
to achieve high sorting efficiencies. Experiments revealed fractionation
of nanobeads from microbeads in the optimized device with high sorting
efficiencies, and protein crystals were sorted into submicrometer
size fractions as desired for future serial femtosecond crystallography
experiments
Additional file 1: of Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography
Figure S1. Schematics of the short-time-point mixing injector. Figure S2. Selected views of the CEF binding site in the BlaC shard crystals including simulated annealing omit maps. Figure S3. Structural details, and simulated annealing omit maps, shard crystal form, subunit B (stereo representation, from 30 ms to 2 s). Figure S4. Structural details and simulated annealing omit maps, shard crystal form, subunit D (stereo representation, from 30 ms to 2 s). Figure S5. Structural details, and simulated annealing omit maps, needle crystal form (stereo representation, from 30 ms to 2 s). Figure S6. Backside view of the catalytic cleft of BlaC in the shard crystal form, structural details and simulated annealing omit maps (stereo representation, selected time points). Figure S7. 2mFo-DFc electron density in the catalytic clefts of BlaC in the shard crystal form (stereo representation, from 30 ms to 2 s). Figure S8. 2mFo-DFc electron density and structural details in the catalytic clefts of BlaC in the needle crystal form (stereo representation from 30 ms to 2 s). Figure S9. Details in the catalytic cleft of subunit B in the shard crystal form at 500 ms including the stacked CEF, 2FoFc maps, and simulated annealing omit maps (stereo representation). Figure S10. The catalytic cleft of BlaC, further details, including a difference map between the 500 ms and 100 ms time points. Figure S11. Crystal packing in shards and needles. Figure S12. Dynamic light scattering results. Table S1. B-factors for CEF species observed in the shard crystals at different time delays. (PDF 1646 kb