Finding Perfect Protein Crystals
Protein crystallography is an important tool for investigation of biological molecules. Protein molecules can crystallize under certain conditions, forming regular lattices composed of multiple copies of the same molecule. When such a crystal is irradiated by a photon beam (x-ray), the photons scatter from the atoms and concentrate in sharp intense spots.
The molecular structure can be determined by analysis of the intensities and positions of the diffraction spots. Co-crystallization of interacting biomolecules or a biological molecule and a chemical drug helps elucidate the protein’s detailed function or find the inhibitor of this function.
Because of the complexity of such experiments, many structural studies require screening hundreds of samples until the crystal with the best resolution is identified.
Traditionally, the rate-limiting step of crystallography studies was a manual sample-loading step. Precise manipulations are required to extract the protein crystal from a liquid nitrogen storage container with special forceps and to place it in the exact position where the crystal can interact with the x-ray beam.
“Automated mounting was a key step in developing fully automated sample screening,” says Clyde Smith, Ph.D., senior staff scientist, Stanford Radiation Laboratory. “Without this manual step, remote data collection also became possible. Now our collaborators all over the world are able to visualize and collect the data stream via a computer interface.”
Stanford Automated Mounter is a robotic arm capable of locating the samples under liquid nitrogen, extracting the sample from storage cassette, and precisely transferring the crystal to the goniometer, a platform that rotates the crystal exposing it to the x-ray beam from various angles.
The robot uses a set of coordinates to find the sample location within the liquid nitrogen dewar. It is also equipped with fast response sensors, preventing it from bumping into the hardware. Periodic calibration ensures precision operation with no sample loss.
“Structure determination of RNA polymerase, a large and complex enzyme, was made possible only by automation of the crystallography process,” says Dr. Smith.
“The team had to screen several hundred samples to find a single crystal with required resolution. Manual screening on this scale is simply not practical.” To date, over 300,000 samples have been screened using Stanford Radiation Laboratory’s automated process.