{"id":442,"date":"2013-04-17T15:49:10","date_gmt":"2013-04-17T15:49:10","guid":{"rendered":"http:\/\/blopig.com\/blog\/?p=442"},"modified":"2013-04-18T12:12:01","modified_gmt":"2013-04-18T12:12:01","slug":"viewing-ligands-in-twilight-electron-density","status":"publish","type":"post","link":"https:\/\/www.blopig.com\/blog\/2013\/04\/viewing-ligands-in-twilight-electron-density\/","title":{"rendered":"Viewing ligands in twilight electron density"},"content":{"rendered":"<p>In this week&#8217;s journal club we discussed an excellent <a href=\"http:\/\/journals.iucr.org\/d\/issues\/2013\/02\/00\/wd5191\/index.html\" target=\"_blank\"> review paper <\/a> by E. Pozharski, C. X. Weichenberger and B. Rupp investigating crystallographic approaches to protein-ligand complex elucidation. The paper assessed and highlighted the shortcomings of deposited PDB structures containing ligand-protein complexes. It then made suggestions for the community as a whole and for researchers making use of ligand-protein complexes in their work.<\/p>\n<p>The paper discussed:<\/p>\n<ul>\n<li>The difficulties in protein ligand complex elucidation<\/li>\n<li>The tools to assess the quality of protein-ligand structures both qualitative and quantitative<\/li>\n<li>The methods used describing their analysis of certain PDB structures<\/li>\n<li>Some case studies visually demonstrating these issues<\/li>\n<li>Some practical conclusions for the crystallographic community<\/li>\n<li>Some practical conclusions for non-crystallographer users of protein-ligand complex structures from the PDB<\/li>\n<\/ul>\n<p><strong>The basic difficulties of ligand-protein complex elucidation<br \/>\n<\/strong><\/p>\n<ul>\n<li>Ligands have less than 100% occupancy &#8211; sometimes significantly less and thus will inherently show up less clearly in the overall electron density.<\/li>\n<li>Ligands make small contributions to the overall structure and thus global quality measures , such as r-factors, will be affected only minutely by the ligand portion of the structure being wrong<\/li>\n<li>The original basis model needs to be used appropriately. The r-free data from the original APO model should be used to avoid model bias<\/li>\n<\/ul>\n<p>The following are the tools available to inspect the quality of agreement between protein structures and their associated data.<\/p>\n<ul>\n<li>Visual inspection of the Fo-Fc and 2Fo-Fc maps,using software such as<a href=\"http:\/\/lmb.bioch.ox.ac.uk\/coot\/\" target=\"_blank\"> COOT<\/a>, is essential to assess qualitatively whether a structure is justified by the evidence.<\/li>\n<li>Use of local measures of quality for example real space correlation coefficients (RSCC)<\/li>\n<li>Their own tool, making use of the above as well as global quality measure resolution<\/li>\n<\/ul>\n<p><strong>Methods and results<\/strong><\/p>\n<p>In a <a href=\"http:\/\/scripts.iucr.org\/cgi-bin\/paper?S1744309112044387\" target=\"_blank\">separate publication<\/a> they had analysed the entirety of the PDB containing both ligands and published structure factors. In this sample they demonstrate 7.6% had RSCC values of less than 0.6 the arbitrary cut off they use to determine whether the experimental evidence supports the model coordinates.<\/p>\n<div id=\"attachment_550\" style=\"width: 451px\" class=\"wp-caption alignleft\"><a href=\"https:\/\/i0.wp.com\/blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.59.26.png\"><img data-recalc-dims=\"1\" decoding=\"async\" aria-describedby=\"caption-attachment-550\" loading=\"lazy\" src=\"https:\/\/i0.wp.com\/blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.59.26.png?resize=441%2C653\" alt=\"Figure to show an incorrectly oriented ligand (a) and its correction (b)\" width=\"441\" height=\"653\" class=\"size-full wp-image-550\" srcset=\"https:\/\/i0.wp.com\/www.blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.59.26.png?w=441&amp;ssl=1 441w, https:\/\/i0.wp.com\/www.blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.59.26.png?resize=202%2C300&amp;ssl=1 202w\" sizes=\"auto, (max-width: 441px) 100vw, 441px\" \/><\/a><p id=\"caption-attachment-550\" class=\"wp-caption-text\">An incorrectly oriented ligand (a) and its correction (b). In all of these figures Blue is the  2mF<sub>o<\/sub>DF<sub>c<\/sub> map contoured at 1&sigma; and Green and Red are positive and negative conturing of the mF<sub>o<\/sub>DF<sub>c<\/sub> map at 3&sigma;<\/p><\/div>\n<p>In this publication they visually inspected a subset of structures to assess in more detail how effective that arbitrary cutoff is and ascertain the reason for poor correlation. They showed the following:<\/p>\n<p>(i)   Ligands incorrectly identi\ufb01ed as questionable,false positives(7.4%)<br \/>\n(ii)  Incorrectly modelled ligands (5.2%)<br \/>\n(iii) Ligands with partially missing density (29.2%).<br \/>\n(iv)  Glycosylation sites (31.3%)<br \/>\n(v) Ligands placed into electron density that is likely to<br \/>\noriginate from mother-liquor components<br \/>\n(vi) Incorrect ligand (4.7%)<br \/>\n(vii) Ligands that are entirely unjusti\ufb01ed by the electron<br \/>\ndensity (11.9%).<\/p>\n<p>The first point on the above data is that the false-positive rate using RSCC of 0.6 is 7.4%. This demonstrates that this value is not sufficient to accurately determine incorrect ligand coordinates. Within the other categories all errors can be attributed to one of or a combination of the following two factors:<\/p>\n<ul>\n<li>The inexperience of the crystallographer being unable to understand the data in front of them<\/li>\n<li>The wilful denial of the data in front of the crystallographer in order that they present the data they wanted to see<\/li>\n<\/ul>\n<div id=\"attachment_548\" style=\"width: 1036px\" class=\"wp-caption aligncenter\"><a href=\"https:\/\/i0.wp.com\/blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.56.10.png\"><img data-recalc-dims=\"1\" decoding=\"async\" aria-describedby=\"caption-attachment-548\" loading=\"lazy\" src=\"https:\/\/i0.wp.com\/blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.56.10.png?resize=625%2C243\" alt=\"Figure to show a ligand placed in density for a sulphate ion from the mother liquor (a) and it&#039;s correction (b)\" width=\"625\" height=\"243\" class=\"size-full wp-image-548\" srcset=\"https:\/\/i0.wp.com\/www.blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.56.10.png?w=1026&amp;ssl=1 1026w, https:\/\/i0.wp.com\/www.blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.56.10.png?resize=300%2C116&amp;ssl=1 300w, https:\/\/i0.wp.com\/www.blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.56.10.png?resize=1024%2C398&amp;ssl=1 1024w, https:\/\/i0.wp.com\/www.blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.56.10.png?resize=624%2C242&amp;ssl=1 624w\" sizes=\"auto, (max-width: 625px) 100vw, 625px\" \/><\/a><p id=\"caption-attachment-548\" class=\"wp-caption-text\">A ligand incorrectly placed in density for a sulphate ion from the mother liquor (a) and it&#8217;s correction (b)<\/p><\/div>\n<p>The paper observed that a disproportionate amount of poor answers was derived from glycosylation sites. In some instances these observations were used to inform the biochemistry of the protein in question. Interestingly this follows <a href=\"http:\/\/www.biomedcentral.com\/1471-2105\/5\/69\">observations<\/a> from almost a decade ago, however many of the examples in the Twilight paper were taken from 2008 or later. This indicates the community as a whole is not reacting to this problem and needs further prodding.<\/p>\n<div id=\"attachment_547\" style=\"width: 516px\" class=\"wp-caption alignleft\"><a href=\"https:\/\/i0.wp.com\/blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.52.49.png\"><img data-recalc-dims=\"1\" decoding=\"async\" aria-describedby=\"caption-attachment-547\" loading=\"lazy\" src=\"https:\/\/i0.wp.com\/blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.52.49.png?resize=506%2C391\" alt=\"Figure to show an incomplete glycosylation site inaccurately modeled\" width=\"506\" height=\"391\" class=\"size-full wp-image-547\" srcset=\"https:\/\/i0.wp.com\/www.blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.52.49.png?w=506&amp;ssl=1 506w, https:\/\/i0.wp.com\/www.blopig.com\/blog\/wp-content\/uploads\/2013\/04\/Screen-shot-2013-04-17-at-13.52.49.png?resize=300%2C231&amp;ssl=1 300w\" sizes=\"auto, (max-width: 506px) 100vw, 506px\" \/><\/a><p id=\"caption-attachment-547\" class=\"wp-caption-text\">Figure to show an incomplete glycosylation site inaccurately modeled<\/p><\/div>\n<p><strong>Conclusions and suggestions<\/strong><\/p>\n<p>For inexperienced users looking at ligand-protein complexes from the PDB:<\/p>\n<ul>\n<li>Inspect the electron density map using COOT if is available to determine qualitatively is their evidence for the ligand being there<\/li>\n<li>If  using large numbers of ligand-protein complexes, use a script such as Twilight to find the RSCC value for the ligand to give some confidence a ligand is actually present as stated<\/li>\n<\/ul>\n<p>For the crystallographic community:<\/p>\n<ul>\n<li>Improved training of crystallographers to ensure errors due to genuine misinterpretation of the underlying data are minimised<\/li>\n<li>More submission of electron-density maps, even if not publically available they should form part of initial structure validation<\/li>\n<li>Software is easy to use but difficult to analyse the output<\/li>\n<\/ul>\n","protected":false},"excerpt":{"rendered":"<p>In this week&#8217;s journal club we discussed an excellent review paper by E. Pozharski, C. X. Weichenberger and B. Rupp investigating crystallographic approaches to protein-ligand complex elucidation. The paper assessed and highlighted the shortcomings of deposited PDB structures containing ligand-protein complexes. It then made suggestions for the community as a whole and for researchers making [&hellip;]<\/p>\n","protected":false},"author":14,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","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":[10,14,9,1],"tags":[],"ppma_author":[508],"class_list":["post-442","post","type-post","status-publish","format-standard","hentry","category-groupmeetings","category-howto","category-talks","category-uncategorized"],"jetpack_featured_media_url":"","jetpack_sharing_enabled":true,"authors":[{"term_id":508,"user_id":14,"is_guest":0,"slug":"anthony","display_name":"Anthony Bradley","avatar_url":"https:\/\/secure.gravatar.com\/avatar\/6ebe5584e1527e5886cfbda3ee205d94b11936a2763a3adeda6c06a2702cc2ea?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\/442","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\/14"}],"replies":[{"embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/comments?post=442"}],"version-history":[{"count":91,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/442\/revisions"}],"predecessor-version":[{"id":585,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/442\/revisions\/585"}],"wp:attachment":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/media?parent=442"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/categories?post=442"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/tags?post=442"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/ppma_author?post=442"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}