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Resistant starch is a prebiotic accessed by gut bacteria with specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses Sas6 (Starch Adherence System member 6), which consists of two starch-specific carbohydrate-binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). Here, we present the crystal structures of Sas6 and of RbCBM74 bound with a double helical dimer of maltodecaose. The RbCBM74 starch-binding groove complements the double helical α-glucan geometry of amylopectin, suggesting that this module selects this feature in starch granules. Isothermal titration calorimetry and native mass spectrometry demonstrate that RbCBM74 recognizes longer single and double helical α-glucans, while RbCBM26 binds short maltooligosaccharides. Bioinformatic analysis supports the conservation of the amylopectin-targeting platform in CBM74s from resistant-starch degrading bacteria. Our results suggest that RbCBM74 and RbCBM26 within Sas6 recognize discrete aspects of the starch granule, providing molecular insight into how this structure is accommodated by gut bacteria. 

Serial synchrotron-based crystallography using intense microfocused X-ray beams, fast-framing detectors and protein microcrystals held at 300 K promises to expand the range of accessible structural targets and to increase overall structure-pipeline throughputs. To explore the nature and consequences of X-ray radiation damage under microbeam illumination, the time-, dose- and temperature-dependent evolution of crystal diffraction have been measured with maximum dose rates of 50 MGy s −1 . At all temperatures and dose rates, the integrated diffraction intensity for a fixed crystal orientation shows non-exponential decays with dose. Non-exponential decays are a consequence of non-uniform illumination and the resulting spatial evolution of diffracted intensity within the illuminated crystal volume. To quantify radiation-damage lifetimes and the damage state of diffracting crystal regions, a revised diffraction-weighted dose (DWD) is defined and it is shown that for Gaussian beams the DWD becomes nearly independent of actual dose at large doses. An apparent delayed onset of radiation damage seen in some intensity–dose curves is in fact a consequence of damage. Intensity fluctuations at high dose rates may arise from the impulsive release of gaseous damage products. Accounting for these effects, data collection at the highest dose rates increases crystal radiation lifetimes near 300 K (but not at 100 K) by a factor of ∼1.5–2 compared with those observed at conventional dose rates. Improved quantification and modeling of the complex spatio-temporal evolution of protein microcrystal diffraction in intense microbeams will enable more efficient data collection, and will be essential in improving the accuracy of structure factors and structural models. 

Small-angle X-ray scattering (SAXS) is an increasingly popular technique that provides low-resolution structural information about biological macromolecules in solution. Many of the practical limitations of the technique, such as minimum required sample volume, and of experimental design, such as sample flow cells, are necessary because the biological samples are sensitive to damage from the X-rays. Radiation damage typically manifests as aggregation of the sample, which makes the collected data unreliable. However, there has been little systematic investigation of the most effective methods to reduce damage rates, and results from previous damage studies are not easily compared with results from other beamlines. Here a methodology is provided for quantifying radiation damage in SAXS to provide consistent results between different experiments, experimenters and beamlines. These methods are demonstrated on radiation damage data collected from lysozyme, glucose isomerase and xylanase, and it is found that no single metric is sufficient to describe radiation damage in SAXS for all samples. The radius of gyration, molecular weight and integrated SAXS profile intensity constitute a minimal set of parameters that capture all types of observed behavior. Radiation sensitivities derived from these parameters show a large protein dependence, varying by up to six orders of magnitude between the different proteins tested. This work should enable consistent reporting of radiation damage effects, allowing more systematic studies of the most effective minimization strategies. 

All evidence to date indicates that at T = 100 K all protein crystals exhibit comparable sensitivity to X-ray damage when quantified using global metrics such as change in scaling B factor or integrated intensity versus dose. This is consistent with observations in cryo-electron microscopy, and results because nearly all diffusive motions of protein and solvent, including motions induced by radiation damage, are frozen out. But how do the sensitivities of different proteins compare at room temperature, where radiation-induced radicals are free to diffuse and protein and lattice structures are free to relax in response to local damage? It might be expected that a large complex with extensive conformational degrees of freedom would be more radiation sensitive than a small, compact globular protein. As a test case, the radiation sensitivity of 70S ribosome crystals has been examined. At T = 100 and 300 K, the half doses are 64 MGy (at 3 Å resolution) and 150 kGy (at 5 Å resolution), respectively. The maximum tolerable dose in a crystallography experiment depends upon the initial or desired resolution. When differences in initial data-set resolution are accounted for, the former half dose is roughly consistent with that for model proteins, and the 100/300 K half-dose ratio is roughly a factor of ten larger. 70S ribosome crystals exhibit substantially increased resolution at 100 K relative to 300 K owing to cooling-induced ordering and not to reduced radiation sensitivity and slower radiation damage. 

Proteins are the workhorses of the cell. The shape that a protein molecule adopts enables it to carry out its role. However, a protein’s shape, or 'conformation', is not static. Instead, a protein can shift between different conformations. This is particularly true for enzymes – the proteins that catalyze chemical reactions. The region of an enzyme where the chemical reaction happens, known as the active site, often has to change its conformation to allow catalysis to proceed. Changes in temperature can also make a protein shift between alternative conformations. Understanding how a protein shifts between conformations gives insight into how it works. A common method for studying protein conformation is X-ray crystallography. This technique uses a beam of X-rays to figure out where the atoms of the protein are inside a crystal made of millions of copies of that protein. At room temperature or biological temperature, X-rays can rapidly damage the protein. Because of this, most crystal structures are determined at very low temperatures to minimize damage. But cooling to low temperatures changes the conformations that the protein adopts, and usually causes fewer conformations to be present. Keedy, Kenner, Warkentin, Woldeyes et al. have used X-ray crystallography from a very low temperature (-173°C or 100 K) to above room temperature (up to 27°C or 300 K) to explore the alternative conformations of an enzyme called cyclophilin A. These alternative conformations include those that have previously been linked to this enzyme’s activity. Starting at a low temperature, parts of the enzyme were seen to shift from having a single conformation to many conformations above a threshold temperature. Unexpectedly, different parts of the enzyme have different threshold temperatures, suggesting that there isn’t a single transition across the whole protein. Instead, it appears the way a protein’s conformation changes in response to temperature is more complex than was previously realized. This result suggests that conformations in different parts of a protein are coupled to each other in complex ways. Keedy, Kenner, Warkentin, Woldeyes et al. then performed X-ray crystallography at room temperature using an X-ray free-electron laser (XFEL). This technique can capture the protein’s structure before radiation damage occurs, and confirmed that the alternative conformations observed were not affected by radiation damage. The combination of X-ray crystallography at multiple temperatures, new analysis methods for identifying and measuring alternative conformations, and XFEL crystallography should help future studies to characterize conformational changes in other proteins.