Since our last blog post, we've carried out an x-ray diffraction experiment with one of our protein crystals. We were lucky that the protein crystal yielded high quality diffraction data, and from this data we were able to solve the first-ever crystal structure of a protein designed by Foldit players—a near-exact match to the designed structure! Below we explain a bit more about x-ray diffraction. In a later post, we'll examine the final structure in more detail.
First, the protein crystal is harvested from the drop using a small loop of nylon, about 0.3 mm across. Protein crystals are often very fragile, so looping the crystal requires a steady hand (i.e. optimal coffee dosage). Even in the loop, the crystal is still immersed in an aqueous solution, with the surface tension of the water helping to keep the crystal in the loop. The loop is rapidly submerged in liquid nitrogen, at a temperature of about -200ºC, which quenches most of the thermal motion of molecules in the crystal.
Once frozen, our looped crystal is mounted on a robotic arm that positions the loop in the path of an x-ray beam. During x-ray exposure, the crystal is kept under a steady stream of cold nitrogen gas to limit temperature increases in the crystal. X-rays have a high energy, and a protein crystal can only endure so much exposure to x-rays before it starts to degrade. The protein lattice could disintegrate from the increased thermal motion of individual protein molecules, or else the x-rays could trigger chemical reactions within the protein, distorting its structure.
X-rays are simply a type of electromagnetic radiation with a very short wavelength—in this case about one angstrom. In an x-ray diffraction experiment, it's important that all radiation has exactly the same wavelength and is focused into a very narrow beam. With our crystal mounted in the path of the x-ray beam, an x-ray detector is positioned behind the crystal, and measures incident x-rays after they strike the crystal and are diffracted by electrons of the protein molecules within. Because of the regular arrangement of atoms in the protein crystal, diffracted x-rays undergo constructive interference in particular directions. This occurs when two equivalent "slices" of the crystal are oriented to coincide with the wavelength of the x-rays. Wherever constructive interference occurs, the detector registers an especially intense signal, shown as a dark spot on the image below. Taken together, these spots comprise a diffraction pattern.
Above is an x-ray diffraction pattern from a protein crystal. In the inset at the right, we can see that some spots seem to have duplicates which are slightly offset. This indicates that there are actually two identical crystals in the path of the x-ray, lying in slightly different orientations. Most likely, the crystal cracked in two during freezing. Fortunately, the image-processing software we use is sophisticated enough to correct for this issue.
The spacing and position of spots is governed by the size and shape of the crystal’s unit cell, the repeating unit that makes up the crystal. The intensity of each spot is determined by the distribution of electrons within the unit cell (i.e. the positions of atoms in the protein). Every atom of the unit cell contributes to each spot in the diffraction pattern. If you could change the electron density around just one atom of your crystallized protein, this would alter the intensity of every spot in the diffraction pattern!
Notice that spots farther from the center of the detector tend to be less intense. More distant spots contain higher resolution data about the electron density of the protein. If we adjust the contrast of this image, we can discern spots close to the edge of the detector. This protein diffracts x-rays to a resolution limit of 1.20 Å! In an electron density map derived from these diffraction patterns, we should be able to distinguish the positions of individual atoms.
If the crystal is rotated relative to the x-ray beam, then we would observe another diffraction pattern, as the new orientation produces constructive interference in different directions. We typically measure a new diffraction pattern at rotation intervals of 0.5 degrees, eventually rotating the crystal a total of 180 degrees (sometimes less for highly-symmetric crystals) to collect a complete dataset. This dataset was collected with a state-of-the-art detector that can measure individual photons; collecting a full dataset takes no more than a few minutes. In the early days of protein crystallography, it could take a whole day to collect a complete dataset!
The processing and interpretation of a these x-ray diffraction patterns is a complex, technical procedure, and we won't go into it here. But suffice it to say, this x-ray diffraction data revealed a full, high-resolution crystal structure of this Foldit player-designed protein!
Congratulations to Waya, Galaxie, and Susume who contributed to this solution in Puzzle 1297! All players should check out Puzzle 1384 to explore the refined electron density map from this data, and see if you can fold up the protein sequence into its crystal structure! We'll follow up later with a more detailed comparison of the designed model and the final crystal structure.( Posted by bkoep 172 3398 | Tue, 05/30/2017 - 04:59 | 4 comments )