Structural Biology Services
Atomic resolution structures of three-dimensional structures of proteins in their unbound states or when bound to other proteins, like antibodies, or bound to small molecule ligands.
Purified and monodisperse protein preparations are screened for conditions that provide large enough crystals (>50 microns). These crystals are frozen in liquid nitrogen and analyzed at synchrotron sources to screen for crystals showing high resolution diffraction. The diffraction data is processed and a model of the protein at atomic resolution is obtained.
Primary Crystal Screening
Identification of promising crystallization conditions for protein of interest using 800-1000 conditions.
Optimization Screens
Optimize promising conditions identified from primary screens to obtain crystals.
Protein Modification
If promising conditions are not identified with the intact preparations of proteins, then the protein can be modified by either in situ proteolysis or lysine methylation. The modified protein can then be screened.
Protein-Ligand Complexes
Structures of protein-ligand complexes can be obtained by crystallizing the protein in the presence of the ligand (co-crystallization) or by soaking the ligand with crystals of the apo-protein.
X-ray Data Collection
Crystals obtained from the screens are frozen in liquid nitrogen and exposed to x-rays at synchrotron sources and the diffraction data is collected.
Structure Determination
X-ray diffraction data obtained are processed to obtain an electron density map. The electron density map together with the known protein sequence is used to obtain the three-dimensional structure of the protein.
Instrumentation
Formulatrix NT8 Crystallization Robot
The NT8 robot can be used to set up sitting drop crystallization plates under 96 different conditions. This can be used to screen 3 different protein samples on a single crystallization plate.
Rock Imager 1000
The Rock Imager 1000 provides automated imaging of crystallization plates on a pre-programmed schedule.
Frequently Asked Questions
Answers to some common questions on protein structure determination.
The primary requirement for crystallizing a protein or protein complex is pure protein. If diffracting crystals are obtained, a known structure of a homologous protein would facilitate structure determination.
Primary crystallization screening involves incubating the protein under conditions varying different physicochemical parameters such as concentration of precipitants, concentration of salts, pH, and temperature. We start by screening the protein against 800-1000 popular crystallizing conditions.
Optimization screening involves varying physicochemical parameters around the promising conditions that have been identified from the primary screens. For example, if 20% PEG 8000 and 01. M Tris-HCl (pH 7.5) are identified as promising conditions that produce crystals, then optimization screens would be set up varying the PEG 8000 from 10 – 30% and the pH would be varied from 7.0 – 8.0.
Primary crystallization screening would require about 2-3 mg of purified protein. The protein should be 95-98 % pure as analyzed on Coomassie-blue stained gel. Another 2-3 mg of protein would be required for optimization screens and for growing crystals that will be sent to the synchrotron beamlines for data collection