![]() These proteins readily form crystals that are large-too big for MicroED and SFX experiments using liquid injectors-but without crystal growth optimization sometimes exhibit pathologies. MicroED has already allowed rapid structure solution from several peptide fragments that resisted solution using microfocus synchrotron sources in spite of many months of crystal optimization ( Supplementary figures 2–4), and the approach could be used for samples where large crystals exist but standard crystallographic methods fail, owing to crystal imperfections.ĢIn all cases the test set comprises approximately 5% of the unique reflections, where possible chosen to match that of the deposited data for the MR search model.įragmentation was tested on eight proteins: lysozyme, TGF-βm:TβRII, xylanase, thaumatin, trypsin, proteinase K, thermolysin, and a segment of the protein tau ( Figure 2, left column). This was true even without further optimization of the growth conditions for crystals formed by various macromolecules of molecular weights between 0.7 kDa and 34.6 kDa, and solvent contents ranging from 30% to 60% with a range of unit cell dimensions and space groups ( Table 1). Breaking of large crystals by physical means produced fragments that appeared crystallographically homogeneous and yielded diffraction data at atomic resolution, void of the above artifacts. These problems are traditionally overcome by modifying the crystallization conditions to optimize crystal growth and quality a process that can be tedious and labor intensive, particularly when crystal pathologies do not become apparent until data processing. In the case of the amyloid-forming peptide of tau (VQIVYK), what appeared to be large crystals were in fact crystal bundles that produced low resolution powder-like diffraction. In many cases, the untreated crystals were mosaic, yielded diffraction patterns with multiple lattices, or were otherwise not suitable for standard crystallographic experiments ( Figure 2, Supplementary figures 1–9). Delicate samples may benefit from gentler fragmentation by vortexing, while harsher methods such as pipetting and ultimately sonication are required to break more robust crystals. Here we show that sonication, vigorous pipetting, or vortexing can be used to break large imperfect crystals into small, single-crystal fragments that are suitable for data collection and atomic structure determination ( Figure 2). This is because large crystals can clog up the nozzle of certain SFX sample delivery systems 6, 7, while in MicroED the large electron scattering cross-section implies that absorption extinguishes diffraction when the sample is too thick 8. In fact, in both these techniques large crystals can prove problematic. These methods can yield structures to resolutions better than 1 Å from crystals that are significantly smaller than those which are required for standard crystallography: ~10,000× smaller in volume for XFEL and ~1,000,000× smaller for MicroED. #Serial box shot 3d 3.6 serial number no download serialMany such defects primarily affect larger crystals, and small crystals may yield superior data quality where diffraction is not limited by the number of diffracting unit cells in the crystal 3.įor these reasons, methods such as serial femtosecond crystallography (SFX) 4 at an X-ray free-electron laser (XFEL) and the electron cryomicroscopy (cryoEM) method microelectron diffraction (MicroED) 5 are actively being developed. Depending on the nature and degree of disorder between the mosaic blocks, an imperfect crystal may exhibit a plethora of pathologies, which may hamper subsequent data reduction, limit the resolution of the final model, and can prevent structure determination altogether ( Supplementary figures 1–9). These mosaic blocks have a finite size, are misaligned with respect to each other, and may be composed of unit cells with different dimensions 2. In the mosaic model, a real, imperfect crystal is composed of several small but well-ordered blocks 1. In reality, crystals of biological material are not perfect. Large and perfect crystals are desirable for structure determination because they yield a strong signal over background. ![]()
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