CHESS DMR-0936384 2013-03-18_1

Breaking the Radiation Damage Limit with Cryo-SAXSJoel Brock, Cornell University, DMR 0936384

Molecular envelopes from nanoliter volumes.

Meisburger, S. P., Warkentin, M., Chen, H., Hopkins, J. B., Gillilan, R. E., Pollack, L., & Thorne, R. E. Breaking the radiation damage limit with Cryo-SAXS. Biophysical Journal 104, 227-236 (2013)

Intellectual Merit: What if you could capture molecules in their solution state, while preventing them from moving during the SAXS experiment? To find out if such an approach could reduce radiation damage in SAXS, Lois Pollack and Rob Thorne's research groups at Cornell and MacCHESS scientist Richard Gillilan borrowed a technique from crystallography called cryo-cooling. The goal of this technique is to cool the solution quickly enough that water forms a glass-like solid, rather than crystalline ice. To make cryo-cooling work for SAXS, they had to leap over several technical hurdles. Unlike crystallography, for SAXS the scattering of the buffer solution must be carefully measured and subtracted to obtain the scattering from the macromolecule alone. Cryo-cooling was thought to be unsuitable for SAXS because it can cause irreproducible formation of small amounts of ice or distortions in the sample shape that would make background subtraction impossible. The Cornell team used the G1 end station at CHESS to show that adding to their solutions sufficient amounts of a chemical similar to anti-freeze, PEG 200, small microliter-sized droplets could be rapidly cooled without forming ice. Molecular envelopes and structures could then be readily obtained.

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Apparatus and method for obtaining SAXS profiles from solution samples at 100 K with a specimen cell using capillary action instead of windows for containment.

Broader Impacts: The use of cryocooling has the potential to eliminate many difficulties associated with room temperatureSAXS experiments. Because vitrified sam-ples are much more radiation tolerant, they may yield much larger integrated signal to noise with no concerns about damage. Samples that spontaneously aggregate or otherwise degrade with time can be frozen immediately after manufacture and stored indefinitely, eliminating concerns about long-term stability. Dramatically reduced sample volume requirements will facilitate mass screening of solution conditions for their effects on molecular structure or association and combinatorial binding assays to, e.g., elucidate pathways for macromolecular complex formation. Short turnaround times will result from exploiting the existing infrastructure for high throughput crystallography, including mail-in facilities that employ automated sample handling and full brightness, for rapid turnaround data collection. In the same way that cryo-MX has transformed atomic resolution studies, cryo-SAXS is poised to transform low-resolution studies of macromolecular structure and function.

Meisburger, S. P., Warkentin, M., Chen, H., Hopkins, J. B., Gillilan, R. E., Pollack, L., & Thorne, R. E. Breaking the radiation damage limit with Cryo-SAXS. Biophysical Journal 104, 227-236 (2013)

Breaking the Radiation Damage Limit with Cryo-SAXSJoel Brock, Cornell University, DMR 0936384

CHESS DMR-0936384 2013-03-18_3

Sorting proteins to the right address at the cellular post office

Joel Brock, Cornell University, DMR 0936384

Crystals (top) and SAXS analysis of exomer solution structures indicating flexibility.

B. C. Richardson, J. C. Fromme, “The Exomer Cargo Adaptor Features a Flexible Hinge Domain,” Structure 21 (3) 486–492 (2013).

Intellectual Merit: How do eukaryotic cells (fungi to mammals to plants) take random mixtures of membrane-bound proteins and sort then into their appropriate vesicle carriers, when the constituent proteins are arranged more as a jumble on the floor than as an orderly assembly line, yet the vesicles are created by highly ordered protein cages? The answer lies in flexibility, so concludes a new study conducted at CHESS beamlines A1 and F2 by postdoctoral associate Brian Richardson and assistant professor Chris Fromme of Cornell University. Recently published in Structure, they combined X-ray crystallography with small-angle X-ray scattering (SAXS) to identify an inherent structural flexibility in one such cargo sorting adaptor, exomer. Exomer is a yeast cargo sorting adaptor that identifies a set of proteins requiring delivery to the plasma membrane at specific places and times in the cell cycle, and pulls them out of the general sorting pool into vesicles for regulated delivery. By solving the crystal structure of the core complex with one of the four cargo-binding proteins, Bch1, Richardson and Fromme showed that two copies of Bch1p are joined together by Chs5 at one end. Pairing this static crystal structure with SAXS data collected on the complex floating free in solution, they further showed that this arrangement allows exomer to open and close akin to a clamshell, with the tips likely at the membrane surface.

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SAXS Analysis of Exomer Solution Structure Indicates Flexibility, shown for assembled Chs5/Bch1 structures indicated.

B. C. Richardson, J. C. Fromme, “The Exomer Cargo Adaptor Features a Flexible Hinge Domain,” Structure 21 (3) 486–492 (2013).

Broader Impacts: Eukaryotic cells, ranging from fungi to mammals to plants, are definedby their complex assortment of internal mem-brane-bound organelles, such as DNA-containing nuclei, energy-producing mito-chondria, and many others. This allows them(us!) to keep important but incompatible activities separate, but it comes at a price: the proteins required for each organelle’s fun-ction, and the proteins destined for the out-side of the cell (the plasma membrane), must be sorted to their separate destinationsby a dedicated organelle, the Golgi appara-tus. Failure to sort proteins into membrane vesicles targeted to the correct destinations leads to a broad spectrum of neurological and other disorders. A new study conducted at CHESS end stations A1 and F2, by postdoctoral associate Brian Richardson and assistant professor Chris Fromme, combines X-ray crystallography with small-angle X-ray scattering (SAXS) to identify an inherent structural flexibility in one such cargo sorting adaptor, exomer. Combined with previous CHESS results, the group developed a model in which three pivots in exomer allow it to adapt to a flexible, diverse membrane environment.

Sorting proteins to the right address at the cellular post office

Joel Brock, Cornell University, DMR 0936384