“hands-off” Screening with TOPAZ ChipsFor the purposes of testing both crystallization behavior and capability of the “tray goniometer”, chips were built to the same specifications as standard commercial TOPAZ chips: a 4mm layer of silicone elastomer (PDMS) mounted on a 1mm thick plastic carrier. This was not a “diffraction-capable” chip as described above.Below are birefringent salt (left) and Lysozyme crystals (right) using polarizing filters. Diffraction data were collected at room temperature at beamline 8.3.1 at the ALS. The inner ring is background scatter from PDMS at ~7A. The second ring is water scatter. The small unit cell of the salt results in a few very strong spots while the larger unit cell in a protein crystal results in hundreds of much weaker diffraction spots. The crystals were not rotated in these images. The incident x-ray wavelength was swept from 0.98Å to 1.24Å to reveal more spots and allow measurement of the mosaic spread from a single image.

Hardware for in situ data collectionThe “tray goniometer” can be readily installed and removed without significant modification to the existing PX endstations at Beamlines 8.3.1 and 12.3.1 at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory (LBNL). The device occupies the space immediately “down beam” from the conventional crystal mount, so there is space for it in most any PX beamline. The Marzhauser motorized Microscope stage will accept SBS-compliant trays or most any crystallization tray that can be turned on it side.  A mirror mounted to the frame reflects the image of the crystal into the optical microscope. This arrangement is parallax-free and the crystal is viewed by the microscope from the “point of view” of the x-ray beam. 

Collection of in situ Diffraction Data from Crystals Grown in TOPAZ® Crystallization Chips

 Andrew P. May1, Ken Frankel2 , George Meigs2, Cesar Luna-Chavez2, John Tainer2,4, Brian Fowler1, James M. Holton2,3

Conclusions

Microfluidic devices for in-chip diffraction data collection have been developed.

Diffraction patterns from completely undisturbed crystals have been collected using a “tray goniometer” at ALS beamlines 8.3.1 and 12.3.1.

High resolution data can be collected directly from crystals located within the chip.

TOPAZ chips have been transported to the synchrotron via FedEx without any signs of damage to the crystals.

The mosaic spread of crystals grown in TOPAZ chips is extremely small.

Future Investigations

Work is underway to collect data from a variety of protein crystals of different morphologies and symmetries to characterize the performance of the chips for in situ data collection.

At present, a new thin chip is under development to reduce the amount of material on the carrier base. Absorption of x-rays by the chip materials and crystallization solution limits the effective resolution of the data that can be collected in situ. The amount of absorption by the new thin chip will be comparable to that of a conventional loop mount.

We will develop methods to overcome the physical limitations imposed by a 2-D array of samples. Options under investigation are collecting data from more than one crystal and using the polychromatic Laue diffraction technique to take advantage of the extremely low mosaic spread of undisturbed crystals.

A rotation axis will be added to the “tray goniometer” so that diffraction data to be collected over a range of up to 120°. 

Cryo-cooling of crystals in the chip is being developed.

1 Fluidigm Corporation, 7100 Shoreline Court, South San Francisco, California2 Lawrence Berkeley National Laboratory, Berkeley, California3 University of California, San Francisco, California4 The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California

TOPAZ Diffraction-capable ChipsDiffraction-capable chips have been designed with different protein to reagent ratios and protein chamber volumes ranging from 0.7 nl to 9 nl. Fabrication processes are being developed to build these chips so that the total chipthickness (all components) is less than 200 μm.

TOPAZ X.96 chips test X protein samples against 96 conditions. For example, the 1.96 chip tests <1.4 µL of protein against 96 different crystallization conditions. Similarly, the 4.96 chip tests 4 samples (each 1.4 µL) against 96 conditions.

We have recently developed a 8.96 chip that tests 8 samples (1.4 µl each) against 96 reagents. The 8.96 chip reduces the number of steps required to set up multiple samples in parallel. This enables the use of a parallel processing strategy to identify crystallization earlier in the structure determination process.

TOPAZ® X.96 Screening Chips

ABSTRACT

We have developed an improved microfluidic device for directly interrogating crystallization experiments with synchrotron X-radiation without removing the crystals from the tray. Identification of conditions for growth of x-ray diffraction quality crystals continues to be a limitation in macromolecular crystallography. This is a particular problem in the case of samples that can only be prepared in limited quantities, such as membrane proteins and multi-component macromolecular complexes.

Fluidigm Corporation has addressed this problem by developing and commercializing the TOPAZ® system for protein crystallization. Crystallization in TOPAZ chips is effected through microfluidic free-interface diffusion (mFID). Current commercially available TOPAZ chips provide the means to screen 96 crystallization conditions using a total of ~1 µl sample.

We describe here new technologies to address the next problem: crystal mounting. Removing crystals from the chip (or indeed any crystallization setup) for diffraction experiments is time consuming and risks damaging the crystal in the process. We have designed and fabricated prototype microfluidic devices based on commercially available TOPAZ crystallization chips that can be mounted directly in an X-ray beam for in situ diffraction experiments. A prototype “tray goniometer” device for performing the in situ diffraction experiments has been constructed and tested at beamlines 8.3.1 and 12.3.1 at the ALS.

Preliminary results are encouraging. Salt can easily be distinguished from protein. TOPAZ chips can be transported to the synchrotron via FedEx without damage to the crystals, and mosaic spreads are typically < 0.02°.

200 μm 200 μm

CarrierChip

Chip bottom layer

Hydration Lid

Hydration lidChip

Chip bottom layer

X-rays

X-rays X-rays

200

μm

salt protein

Conventionalmounting loop

x-rays

microscope

mirror crystalpinhole