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I am very new to protein crystallography and visualization. I have been asked to take photos through an optical microscope of the lysozyme powder directly as-is from the supplier. I suspended some of the powder in both paraffin and glycerol (10 mg/mL & 20 mg/mL for each) to observe the crystal form without solubilizing it.

I'm not sure what it is I'm looking at, however, in all of the suspensions I prepared, the particles look very different from literature images of both lysozyme and salt crystals. The image I included is a 10 mg/mL suspension in paraffin but is essentially what I'm seeing in all cases. A mixture of large chunks with some smaller flakes.

I am wondering if anyone with protein crystal experience can tell me what this form I'm seeing is? And perhaps any further suggestions on how to visualize the sample without any alteration. Thank you Lysozyme powder in paraffin oil

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  • $\begingroup$ They do not look like single crystal pieces, but I'm not sure that they are not polycrystalline in nature. $\endgroup$
    – Jon Custer
    May 18, 2023 at 19:25

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The typical protein crystal does not withstand mechanical stress equally well than e.g., the ones of table salt, or refined sucrose. Hence the varying shapes of grains.

Photos about nice protein crystals by shape (habit) & extinction (check if you can use linearly polarized light / "crossed Nicols") often are recorded while they still are in their mother liquor (to grow them e.g., by sitting drop, hanging drop, or in capillary [see e.g. here] to mention some techniques) typically grown for in-house/beamline X-ray diffraction analysis. Here, you want them to be representative of your sample (e.g., in chemical composition, and crystalline phase), and yet good enough for subsequent data collection and data processing.

The commercial (perhaps microcrystalline) powder you have access to, is prepared with the primary intent to offer you access to a sample chemically (by molecular composition) pure enough. That is, concentrations of remaining solvent of reaction / purification, unwanted ions of heavy metals are below an (often arbitrary) threshold set. If there once were crystals, they now are broken in a more or less random way while passing sieves, being scrapped-off from filters, dried (think about solvent molecules like water which have to leave the solid), filled into a bottle which upon transport is shaken / crystals bounced against each other. This reduces your chances to meet a crystal with prominent cleavage planes. Instead, you find rough and conchoidal surfaces. In addition, the size of the grains may follow a more or less broad grain distribution, too.

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    $\begingroup$ Your first sentence isn't clear to me, do you mean protein crystals are just as bad at withstanding mechanical stress as salt/sugar are, or that protein crystals are worse at withstanding mechanical stress? $\endgroup$
    – llama
    May 17, 2023 at 13:53
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    $\begingroup$ Generally speaking, @llama, protein crystals are much more fragile than crystals of ionic salts or small molecules. They also invariably contain a large proportion of water, so protein crystals generally cannot endure being very long removed from their mother liquor to a dry environment. $\endgroup$ May 17, 2023 at 18:52
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    $\begingroup$ It often is easier to damage a protein crystal, than the ones of small molecules. It then is more difficult to record their shape/dimensions, perform an absorption correction; mechanically damaged crystals often feature broader/more often partially split/overlapping individual X-ray diffraction peaks which can render them unsuitable for single crystal XRD. You still have powder XRD as a complement which by today very well includes proteins (beside the sample a question of equipment & accessing a source brilliant enough). See e.g. Margiolaki (doi.org/10.1524/9783486992540-004, OA). $\endgroup$
    – Buttonwood
    May 17, 2023 at 19:18
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    $\begingroup$ Sometimes, protein gets sold in a freeze-dried formulation. In that case, you don't expect crystals, and sometimes the protein will denature (which is find for some applications, such as protein standards in SDS PAGE). See e.g. Roy and Gupta $\endgroup$
    – Karsten
    Jun 12, 2023 at 14:38

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