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A Beginner's Guide to Macromolecular Crystallization

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A Beginner's Guide to Macromolecular Crystallization Beginner’s Guide A beginner’s guide to macromolecular crystallization Fabrice Gorrec (MRC Obtaining diffraction- quality crystals is currently the rate-limiting step in macromolecular X-ray Laboratory of Molecular crystallography of proteins, DNA, RNA or their complexes, in the vast majority of cases. Since each Biology, U.K.) sample has different and specific characteristics – which is the reason for wanting to study every single Downloaded from http://portlandpress.com/biochemist/article-pdf/43/1/36/903768/bio_2020_108.pdf by UK user on 22 March 2021 one of them in the first place – crystallization conditions cannot be predicted. Hence, researchers must enable crystal nucleation and growth through experimentation and screening. The size, shape and surface of the sample or complexes of interest are often altered through genetic and biochemical manipulation to facilitate crystallization, based on bioinformatics analyses and trial and error. Pure samples are trialled against a very broad range of crystallization conditions. The currently predominant method to achieve crystallization is sitting drop vapour diffusion with nanolitre- class robotic liquid handlers. Once initial screening yields crystals, further optimization experiments are usually required to obtain larger and diffraction- quality crystals. Background as possible. It should reach a concentration of at least a few grams per litre, which is high for most samples. Macromolecular X- ray crystallography applied to Samples that are not very stable should be trialled crystals of biological molecules enables us to visualize readily after their preparation; some even require low structures of proteins, DNA, RNA and their complexes temperature handling or oxygen depletion during the with near to full atomic resolution (~3.5 Å to less than entire crystallization process. 1 Å resolution). Crystallography’s immense power lies in the fact that it makes visible complex atomic structures, sometimes with hundreds of thousands of atoms. It enables the understanding of biology through Summary box the lens of the laws of chemistry and physics. It is also employed to aid structure- based, rational drug design. • Successful structure determination is usually easier X- ray crystallography as a method has matured the stronger X- ray diffraction is from the crystals, tremendously since its inception in the 1950s at the MRC and should be better than 3.5 Å resolution. Laboratory of Molecular Biology (LMB), the birthplace • Appearance of crystals can be misleading, and of protein crystallography. Through the use of much hence, their true usefulness is best established by brighter X- ray beams at synchrotrons and sophisticated X- ray diffraction. computer programmes, once high- quality crystals • Often, many sample variations and preparations are have been made available, structure determination required for solving novel or complicated crystal has become a mere technicality. In contrast, obtaining structures. crystals has been an increasingly severe bottleneck. • Different types of crystallization screen formulations, Some projects take years before crystals can be obtained, integrating a plethora of suitable chemicals, are if at all. Large proteins and complexes (>200 kDa) are employed to alter the variables determining the particularly difficult to crystallize. crystallization experiments. In order to predict how to crystallize a protein, • When enough sample is available, a large initial what would help most would be details of its atomic screen increases the likelihood of obtaining useful crystals. structure, the very thing that the method tries • Nucleation is often rate limiting for obtaining to obtain. Since each protein has an unavoidably crystals. different structure, we are required to employ • Crystal growth after nucleation is typically affected sophisticated screening procedures to empirically by defects that limit crystal size. find the conditions under which the sample forms • Various seeding techniques can be applied to crystals. Furthermore, a sample to be trialled for overcome the nucleation bottleneck. crystallization needs to be as pure and homogeneous 36 February 2021 © The Authors. Published by Portland Press Limited under the Creative Commons Attribution License 4.0 (CC BY-NC- ND) Beginner’s Guide The basis of crystallography is that a crystal is a The quality of the internal order of the crystals is often nearly perfect periodic three- dimensional arrangement reflected in their appearance, where sharp edges can be of molecules (the ‘lattice’). A crystal amplifies the weak a sign of crystals which will diffract well. However, even scattered X- ray signal from each molecule only in the crystals that look promising have typically high solvent direction where constructive interference between the content (in the order of 50%–70%). Besides, they are diffracted beams occurs. Therefore, larger crystals often, mainly formed through weak interactions such as small but not always, diffract more strongly. The conditions for hydrophobic surfaces, water- mediated, longer- range constructive interference are condensed in Bragg’s law. and flexible electrostatic interactions and sometimes Diffraction patterns are recorded on two- dimensional even single hydrogen bonds. On the one hand, this detectors while the crystal is rotated. Once the intensities is beneficial as the macromolecules are unlikely to be of all the beams have been determined, the three- altered by the binding energies generated during the dimensional electron density function/map can be built crystallization process. On the other hand, the weakness Downloaded from http://portlandpress.com/biochemist/article-pdf/43/1/36/903768/bio_2020_108.pdf by UK user on 22 March 2021 using a number of standard methods to solve the phase of the interactions means that protein crystals have a problem. The phase problem arises because only the mosaic structure (Figure 1b). This so-called mosaicity intensities of the diffracted beams are recorded and not broadens the diffracted beams and lowers their signal- their relative phase angles, which are needed to solve the to- noise ratio on the detector. structure. However, for crystals that diffract beyond 3.5 An even more severe crystal pathology, twinning, Å resolution, standard methods exist to solve the phase can occur when parts of the crystal grow in distinct, but problem, such as molecular replacement (MR; using a pseudo symmetry- related orientations (Figure 1c). The homologous structure), single anomalous diffraction crystal can, therefore, be packed in nearly identical ways using selenomethionine incorporation (SeMet SAD) or to yield the same lattice structure, which can sometimes single/multiple isomorphous replacement (S/MIR, Max lead to an inability to solve the structure. Perutz’ original heavy metal–binding procedure). Macromolecular crystals The sample is the main variable of crystallization The largest dimension of a typical small protein is about 10 nm. A crystal of the same protein, big enough to Samples of interest are generated using molecular be visible under a microscope, is a lattice formed by biology methods. For example, recombinant proteins billions of the protein molecules (Figure 1a). Crystal are produced with expression systems such as bacteria, morphology is dictated by the lattice type and symmetry insect cells or mammalian cells. For successful (https:// it. iucr. org/). crystallization, many samples require the removal of Figure 1. (a) A promising bipyramidal- shaped protein crystal ready to be tested for X- ray diffraction. (b) A highly exaggerated representation of crystal mosaicity (adapted from Phil Jeffrey’s course on X- ray data collection, xray0.princeton. edu/~phil/Facility/). (c) Representation of a possible case of crystal twinning: crystal formed with two pyramid structures in opposite orientations (from Zbigniew Dauter’s course on Twinning in protein crystal, https://slideplayer.com/slide/4983758/). February 2021 © The Authors. Published by Portland Press Limited under the Creative Commons Attribution License 4.0 (CC BY-NC- ND) 37 Beginner’s Guide flexible and small disordered parts, problematic domains solvation of the protein decreases, causing its properties and/or subunits. Aggregation properties may need to be to change, such that hydrophobic interactions on the improved, as rapid, nonspecific aggregation competes surface are accentuated. The effect on precipitation is with the crystallization process. proportional to the salt concentration and its Hofmeister In principle, increasing the rigidity of the sample, ranking. Other types of precipitants, such as polyethylene making it more soluble and stable in the chosen buffer, glycols (PEGs), also compete with solvent molecules on increases the chances of successful crystallization and the protein surface in different ways. Varying the buffer’s also the resulting X- ray diffraction. However, rigidity pH will alter the density of charges on the surface of and simplicity do not systematically produce the best macromolecules, altering both their solubility and outcome. Besides, secondary indicators of sample the possibilities of interactions. Other chemicals can quality, such as stability, purity or amounts available, be employed to affect solubility, but often have more can be misleading. As a result, empirical approach is specific effects that go beyond solubility and are termed Downloaded from http://portlandpress.com/biochemist/article-pdf/43/1/36/903768/bio_2020_108.pdf by UK user
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