Most of the work I do involves comparing diffraction datasets from protein crystals. We often\u00a0have two or more different crystals of\u00a0the same crystal system, and want to spot differences between them. The crystals are nearly isomorphous, so that the structure of the protein (and crystal) is almost identical between the two datasets. However, it’s not just a case of overlaying the electron density maps, subtracting them and looking at the difference. Nor do we necessarily want to calculate Fo-Fo maps, where we calculate the difference by directly subtracting\u00a0the diffraction data before calculating maps.\u00a0By the nature of the crystallographic experiment, no two crystals are the same, and two (nearly identical) crystals can lead to two quite different datasets.<\/p>\n
So, here’s a list of things I\u00a0keep in mind when comparing crystallographic datasets…<\/p>\n
1)<\/strong><\/em> Ensure that the resolution limits in the datasets are the same, both at the high AND\u00a0the low resolution limits.<\/strong><\/em><\/span><\/p><\/blockquote>\n
The High resolution limit.<\/strong> The best known, and (usually) the most important statistic\u00a0of a dataset. This is a measure of the amount of information that’s been collected about the dataset. Higher resolution data gives more\u00a0detail for the electron density. Therefore, if you compare a 3A map to a 1A map, you’re comparing fundamentally different objects, and the differences between them will be predominantly from the different amount of information in each dataset. It’s then very difficult to ascertain what’s interesting, and what is an artefact of this difference.\u00a0As a first step, truncate all datasets at the resolution you wish to compare them at.<\/p>\n
The Low Resolution Limit.<\/strong> At the other end of the dataset, there can be differences in the low resolution data collected. Low resolution reflections correspond to much larger-scale features in the electron density. Therefore, it’s just as important to have the same low-resolution limit\u00a0for both datasets, otherwise you get large “waves” of electron density (low-frequency fourier terms) in one dataset that are not present in the other. Because low-resolution terms are much stronger than high resolution reflections,\u00a0these features stand out very strongly, and can also obscure “real” differences between the datasets you’re trying to compare. Truncate\u00a0all datasets at the same low resolution limit as well.<\/p>\n
Consider the Unit Cell<\/h2>\n
2)<\/strong> Even if the resolution limits are the same, the number of reflections\u00a0in maps can be different.<\/strong><\/span><\/em><\/p><\/blockquote>\n
The Unit Cell size and shape.<\/strong> Even if the crystals you’re using are the same crystal form, no two crystals are the same. The unit cell (the building block of the crystal) can be slightly different sizes and\u00a0shapes between crystals, varying in size by a few percent. This can occur by a variety of reasons,\u00a0from the unpredictable process of cooling the crystal to cryogenic temperatures to entirely stochastic differences from the process of crystallisation. Since the “resolution” of reflections depends on the size of the unit cell, two reflections with the same miller index can have different “resolutions” when it comes to selecting\u00a0reflections for map calculation. Therefore, if you’re calculating maps from nearly-isomorphous but non-identical crystals, consider calculating maps based on an high and a low miller index<\/em><\/strong> cutoff, rather than a resolution cutoff.\u00a0This ensures the same amount of information in each map (number of free parameters).<\/p>\n
Watch for Missing Reflections<\/h2>\n
3)<\/strong><\/em>\u00a0Remove any missing reflections from both datasets.<\/strong><\/em><\/span><\/p><\/blockquote>\n
Reflections can be missing\u00a0from datasets for a number of reasons, such as falling into gaps\/dead pixels on the detector. However, this isn’t going to happen systematically with all crystals, as different crystals will be mounted in different orientations. When a reflection is missed in one dataset, it’s best to remove it from the dataset you’re comparing it to as well. This can have an important effect when the completeness of low- or high-resolution shells is low, whatever the reason.<\/p>\n
Not All Crystal Errors\u00a0are Created Equal…<\/h2>\n
4)<\/em><\/strong>\u00a0<\/em><\/strong>Different Crystals have different measurement errors.<\/em><\/strong><\/span><\/p><\/blockquote>\n
Observation uncertainties\u00a0of reflections will vary from crystal to crystal. This may be due to a poor-quality crystal, or a crystal that has suffered from more radiation damage than another. These errors lead to uncertainty and error in the electron density maps. Therefore, if you’re looking for a reference crystal, you probably want to choose one with as small uncertainties,\u00a0\u03c3(F),\u00a0in the reflections as possible.<\/p>\n
Proteins are Flexible<\/h2>\n
5)<\/strong> Even though the crystals are similar, the protein may\u00a0adopt slightly difference conformations.<\/strong><\/span><\/em><\/p><\/blockquote>\n
In real-space, the\u00a0protein structure varies from crystal to crystal.\u00a0For the same crystal form, there will be the same number of protein copies in the unit cell, and they will be largely in the same conformation. However,\u00a0the structures are not identical, and the inherent flexibility of the protein can mean that the conformation seen in the crystal can change slightly from crystal to crystal. This effect is largest in the most flexible regions of the protein, such as unconstrained\u00a0C- and N- termini, as well as flexible loops and crystal contacts.<\/p>\n","protected":false},"excerpt":{"rendered":"
5 Thoughts For… Comparing Crystallographic Datasets Most of the work I do involves comparing diffraction datasets from protein crystals. We often\u00a0have two or more different crystals of\u00a0the same crystal system, and want to spot differences between them. The crystals are nearly isomorphous, so that the structure of the protein (and crystal) is almost identical between […]<\/p>\n","protected":false},"author":21,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"quote","meta":{"nf_dc_page":"","wikipediapreview_detectlinks":true,"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"ngg_post_thumbnail":0,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[10],"tags":[],"ppma_author":[512],"class_list":["post-2527","post","type-post","status-publish","format-quote","hentry","category-groupmeetings","post_format-post-format-quote"],"jetpack_featured_media_url":"","jetpack_sharing_enabled":true,"authors":[{"term_id":512,"user_id":21,"is_guest":0,"slug":"nick","display_name":"Nick","avatar_url":"https:\/\/secure.gravatar.com\/avatar\/578b36ab386a19b5b0509915b0ab6e7cf2193ce737044a31fd0b3251416f07b1?s=96&d=mm&r=g","0":null,"1":"","2":"","3":"","4":"","5":"","6":"","7":"","8":""}],"_links":{"self":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/2527","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/users\/21"}],"replies":[{"embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/comments?post=2527"}],"version-history":[{"count":10,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/2527\/revisions"}],"predecessor-version":[{"id":2538,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/2527\/revisions\/2538"}],"wp:attachment":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/media?parent=2527"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/categories?post=2527"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/tags?post=2527"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/ppma_author?post=2527"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}