In addition to the workhorse techniques of macromolecular crystallography (MX) and BioSAXS, MacCHESS welcomes experiments that use X-rays in other ways, as well as novel applications of MX and BioSAXS. Have an idea for an off-the-wall experiment? Contact us and talk about it!
MacCHESS: Beyond the usual
MacCHESS offers users an advanced serial microcrystallography experience.
Biological small-angle X-ray solution scattering is a popular technique for obtaining structural information directly from solutions of biomolecules. Scattering profiles obtained this way can yield radius of gyration, molecular weight, maximum dimension, degree of disorder, and even low-resolution electron density. When combined with known models, advanced processing techniques can help assemble complexes from individual domains and make statements about which conformations are likely present in solution. Rapid microfluidic mixing technology now provides a means of watching how solution scattering profiles change on timescales of a few milliseconds or less. If your system may be going through a series of conformational transitions, of changes in flexibility or oligomeric state, then this technique could tell you something important. Contact Richard Gillilan to discuss sample requirements and feasibility.
High Pressure Small Angle X-ray Scattering
Diffuse scattering experiments
Enzyme Dynamics beyond Bragg Diffraction
Nozomi Ando, Ph.D., Assistant Professor of Chemistry & Chemical Biology,
Cornell University, Department of Chemistry & Chemical Biology
X-ray crystallography has proven to be an invaluable tool in structural and mechanistic studies of enzymes. However, our knowledge of how protein dynamics affect function is largely incomplete. Although Bragg diffraction can indicate mobility in a protein structure, it does not contain information on how motions occur in concert. To access this information, we must venture beyond Bragg diffraction into a complex, relatively unexplored region of X-ray data known as diffuse scattering. All proteins are dynamic, even inside crystals. Non-periodic patterns in the lattice arising from correlated motions leads to a cloudy, textured background pattern that is routinely thrown away in conventional crystallography.
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3. Temiz, N.A., and Bahar, I. (2002) Inhibitor binding alters the directions of domain motions in HIV-1 reverse transcriptase. Proteins 49, 61-70.
4. Shen, L., Shen, J., Luo, X., Cheng, F., Xu, Y., Chen, K., Arnold, E., Ding, J., and Jiang, H. (2003) Steered molecular dynamics simulation on the binding of NNRTI to HIV-1 RT. Biophys. J. 84, 3547-3563.
5. Kung, Y., Ando, N., Doukov, T.I., Blasiak, L.C., Bender, G., Seravalli, J., Ragsdale, S.W., and Drennan, C.L. (2012) Visualizing molecular juggling within a B12-dependent methyltransferase complex. Nature 484, 265-269. PMCID: PMC3326194
6. Zhang, Y., and Stubbe, J. (2011) Bacillus subtilis class 1b Ribonucleotide Reductase is a dimangansese(III)-tyrosyl radical enzyme. Biochemistry 50, 5615-5623.
7. Jarrett, J.T., Huang, S., and Matthews, R.G. (1998) Methionine synthase exists in two distinct conformations that differ in reactivity towards methyletrahydrofolate, adenosylmethionine and flavodoxin. Bichemistry 37, 5372-5382.
8. Meisburger S.P., Ando N., "Correlated Motions from Crystallography beyond Diffraction," Accounts of Chemical Research 50(3), 580-583, 2017.
Experiments under pressure in diamond anvil cell (DAC)
How does the diffraction from a single crystal change with pressure?
For these experiments, a protein crystal was mounted into the sample chamber of a DAC and the pressure was increased step by step (0, 200, 400MPa); at each pressure 1-2 diffraction images were collected. Diffraction data were collected at the A1 station at room temperature using a Q-270 detector; the X-ray energy was 19.3keV. To avoid dehydration during mounting of the crystal into the sample chamber of DAC, all crystals were coated with NVH oil. As the sample chamber is small (thickness ~50µm, diameter ~200µm), only small, thin crystals were picked for pressurization. The setup was tested with lysozyme and trypsin crystals and found to work well.
Crane, B. R. (2008). Biochem Soc Trans. 36, 1149-1154.
Huang, Q., Gruner, S. M., Kim, C. U., Mao, Y., Wu, X., & Szebenyi, D. M. 2016. J. Appl. Crystallogr. 49, 149-157.
Pant, K. & Crane, B. R. (2006). Biochemistry 45, 2537-2544.
This technique is under development, and must be done in collaboration with MacCHESS staff.
Contact Qingqiu Huang if interested.
Diffraction from cellulose fibers in plant stems, to determine the crystallinity of the cellulose, for a biofuels study.
Diffraction from crystals in droplets in a capillary, as part of a collaborative serial crystallography project.
Monitoring of radiation damage using Laue diffraction.
Irradiation of virus samples with X-rays, as a test of alternative means of inactivating the virus.
Use of SAXS to study the hydration of gels.
Observing molecular alignment in biofilms
Biofilms are communities of microorganisms that stick to surfaces using films of natural polymers such as polysaccharides. How microorganisms navigate and organize within the polymer is a subject of great importance in both in medicine and industry. X-ray scattering is a sensitive probe of alignment of molecules and the formation of order in soft materials. Using a novel inclined ring support, controlled compression of polysaccharide gels can reveal induced alignment of fibers that has important implications for how bacteria spread within the gel. MacCHESS is experienced with creating novel sample support and manipulation setups integrated with high-sensitivity X-ray scattering detection and strong data processing support software.
David J. Lemon, Xingbo Yang, Pragya Srivastava, Yan-Yeung Luk & Anthony G. Garza. Polymertropism of rod-shaped bacteria: movement along aligned polysaccharide fibers. Scientific Reports 7, Article number: 7643 (2017) doi:10.1038/s41598-017-07486-0.
Users interested in studying gels and other biologically-related soft matter should contact Richard Gillilan to discuss feasibility.