sporte https://scienceblogs.com/author/sporte/feed en New home for Discovering Biology in a Digital World https://scienceblogs.com/digitalbio/2017/10/30/new-home-for-discovering-biology-in-a-digital-world <span>New home for Discovering Biology in a Digital World</span> <div class="field field--name-body field--type-text-with-summary field--label-hidden field--item"><p>Sometime in the next day or two, Scienceblogs will shut down.  We've enjoyed the opportunity to blog here for the past 10+ years. Not to worry, @digitalbio and @finchtalk will continue blogging, but more so from their own site at <a href="https://digitalworldbiology.com">Digital World Biology</a>.  The Scienceblogs posts have been reposted at <a href="https://digitalworldbiology.com/digitalbio/scienceblogs-archive">Digital World Biology's scienceblog archive</a>, and new posts will be at <a href="https://digitalworldbiology.com/blog">Discovering Biology in a Digital World</a>, now at Digital World Biology.</p> <p>Enjoy</p> <p>@digitalbio, @finchtalk</p> <p><a href="https://www.sockeyephotography.com/Travel/Alaska-2017/i-cKzNg72/0/00745eab/M/20170831-008-M.jpg"><img class="alignnone" src="https://photos.smugmug.com/Travel/Alaska-2017/i-cKzNg72/0/00745eab/M/20170831-008-M.jpg" alt="" width="599" height="400" /></a></p> </div> <span><a title="View user profile." href="/author/sporte">sporte</a></span> <span>Mon, 10/30/2017 - 18:46</span> Mon, 30 Oct 2017 22:46:03 +0000 sporte 69989 at https://scienceblogs.com Synbiobeta: The Future is Now https://scienceblogs.com/digitalbio/2017/10/12/synbiobeta-the-future-is-now <span>Synbiobeta: The Future is Now</span> <div class="field field--name-body field--type-text-with-summary field--label-hidden field--item"><p>@synbiobeta concluded it’s #sbbsf17 annual meeting on synthetic biology Oct 5, 2017. The progress companies are making in harnessing biology as a platform for manufacturing and problem solving is world changing.</p> <div style="width: 410px;display:block;margin:0 auto;"><a href="https://www.biotech-careers.org/company-core-activity/synthetic-biology" target="_blank" rel="noopener noreferrer"><img class="wp-image-1251 size-large" src="http://scienceblogs.com/digitalbio/files/2017/10/synbio-around-the-world-400x109.jpeg" alt="" width="400" height="109" /></a> Locations of Synbio Companies </div> <p><strong>What is Synthetic Biology?</strong></p> <p>Synthetic biology is a term that is used to describe the convergence of biotechnology and engineering. The dramatic cost decreases in our ability to read and write DNA (sequence and synthesize), combined with increasing capabilities in automation and informatics has catalyzed a new sub-industry within biotechnology. Synthetic biology utilizes high-throughput combinatorial approaches to quickly evolve living systems to make new drugs, foods, and chemicals, to mention a few. It is the culmination of nearly 50 years of biotechnology discovery and refinement, The new products created by biological systems are poised to tackle many of the world’s greatest problems</p> <p>While the core activities of synthetic biology, gene cloning and constructing plasmids for gene expression have been around for nearly 50 years, synthetic biology capitalizes on technologies that miniaturize and implement processes in massively parallel formats. Miniaturization, be it solid phase or micro/nano container formats, significantly reduces reagent costs. Massively parallel formats increase the numbers of reactions and operations that can run simultaneously from a few hundred, as is done with 96-well plates, to millions and billions, as are now routine in DNA sequencing and DNA synthesis. These capabilities allow researchers to test new designs to optimize enzymes, create specialty proteins, and control gene expression in random and directed ways.</p> <p><strong>The Synbiobeta Meeting</strong></p> <p>The @synbiobeta meeting celebrated its seventh year with more than 600 attendees and 50 exhibiting companies. Most of the presentations were by new companies demonstrating both the current and future impact of synthetic biology on the economy. A major theme being how biological systems will be able to produce materials that are currently produced from petroleum products. Replacing oil’s pervasive presence is an important goal in battling climate change, reducing environmental pollution, and promoting national security.</p> <p>The presentations were organized around themes with several key notes and “fireside” chats that involved interview formats. Jason Kelly, CEO of <a href="https://www.biotech-careers.org/company/ginkgo-bioworks">Ginkgo Bioworks</a> kicked the meeting off by highlighting his favorite Synthetic Biology companies. These included <a href="https://www.biotech-careers.org/company/bolt-threads">Bolt Threads</a>, harnessing spider silk production in bacteria; <a href="https://www.biotech-careers.org/company/impossible-foods">Impossible Foods</a>, working on "cellular agriculture" - the flavor is in the heme;  and <a href="https://www.biotech-careers.org/company/twist-bioscience">Twist Bioscience</a>, leading the way with oligo and gene synthesis. Ginkgo recently agreed to purchase one billion bases of synthesized DNA from Twist, demonstrating the scale of synthetic biology. Kelly also talked about the <a class="ext" href="http://science.sciencemag.org/content/early/2017/08/09/science.aan4187" target="_blank" rel="noopener noreferrer">CRISPR pig <span class="ext"><span class="element-invisible">(link is external)</span></span></a>that recently appeared in Science Magazine. George Church’s group, in collaboration with many others, used CRISPR-based gene editing to remove endogenous retroviruses, which will opens opportunities for future xeno-transplantation. Of course there likely will be issues with immunity to those pesky pig proteins and more CRISPieR pigs will be needed before goal of xeno-transplantation can be fully realized. Kelly closed his talk by discussing how foundry concepts from industrial processes are being applied to biology to emphasize how the convergence of engineering in biotechnology.</p> <p>The remaining talks were organized into themes that emphasize both what is possible in synthetic biology and what is needed to enable those possibilities. Sessions like Biomaterials and Consumer Products, Cell Factories for Biopharmaceuticals and Healthcare, Thought for Food, Innovations for Ocean Sustainability,  Protein is the Killer App, Environmental Application of Synthetic Biology, and the keynotes, had an obvious focus on what is being done with synthetic biology, whereas sessions like the Panel Discussion on the Future of DNA Synthesis, Computation and Synthetic Biology, and Sponsor presentations from <a href="https://www.biotech-careers.org/company/labcyte">Labcyte</a>, <a href="https://www.biotech-careers.org/company/integrated-dna-technologies">IDT</a>, <a href="https://www.biotech-careers.org/company/twist-bioscience">Twist</a>, <a href="https://www.biotech-careers.org/company/genscript">GenScript</a>, had focus on the enabling technologies needed to do synthetic biology.</p> <p>The next blogs will share details from these sessions. In the mean time check out the companies enganged in <a href="https://www.biotech-careers.org/company-core-activity/synthetic-biology">synthetic biology</a>.</p> </div> <span><a title="View user profile." href="/author/sporte">sporte</a></span> <span>Thu, 10/12/2017 - 08:45</span> Thu, 12 Oct 2017 12:45:08 +0000 sporte 69988 at https://scienceblogs.com Understanding the CRISPR Cas9 system https://scienceblogs.com/digitalbio/2016/09/18/understanding-the-crispr-cas9-system <span>Understanding the CRISPR Cas9 system</span> <div class="field field--name-body field--type-text-with-summary field--label-hidden field--item"><p>On Sept. 30th, I'm going to be co-presenting a Bio-Link webinar on Genome Engineering with CRISPR-Cas9 with Dr. Thomas Tubon from Madison College.  If you're interested, <a href="https://events-na2.adobeconnect.com/content/connect/c1/916595931/en/events/event/shared/default_template_simple/event_registration.html?sco-id=983023884&amp;_charset_=utf-8">Register here</a>.  Since my part will be to help our audience understand the basics of this system, I prepared a short tutorial with <a href="http://digitalworldbiology.com/products/molecule-world">Molecule World</a> .  Enjoy!</p> <p><strong>A Quick CRISPR Tutorial</strong></p> <ol><li>Go to the Digital World Biology <a href="http://digitalworldbiology.com/collection/crispr">CRISPR Structure Collection</a>.</li> <li>Download the second item in the list, 5F9R, by clicking the link in the Download structure column.</li> <li>Identify the three components of the CRISPR - Cas system:  The Cas9 protein, the guide RNA, and the target DNA.</li> <li>Use the camera button to capture an image of each component and paste it in your lab notebook.</li> </ol><p style="padding-left: 60px;">To view the individual components, find and highlight the different kinds of sequences one at a time.</p> <p style="padding-left: 60px;">Use a combination of hiding structures and changing the coloring and drawing styles to make it easier to distinguish the parts.</p> <p style="padding-left: 60px;">You may want to zoom in and zoom out to see how the nucleic acids are positioned within the protein.</p> <p style="padding-left: 60px;"><em>Suggestion</em> - one nice way to view the structure is to select the protein, show all the residues, make it spacefill, and color it neutral.  Lock the chains.</p> <p style="padding-left: 60px;">Then select the nucleic acids to highlight them and change the coloring and drawing styles so you can see how they're positioned in the protein.</p> <p style="padding-left: 60px;"><img class="size-large wp-image-1228 alignnone" src="http://scienceblogs.com/digitalbio/files/2016/09/CRISPR_tutorial_1-400x303.png" alt="crispr_tutorial_1" width="400" height="303" /></p> <p style="padding-left: 30px;">5.  When, you’re done exploring, use Reset View to restore the original drawing and coloring styles.</p> <p style="padding-left: 30px;"><img class="size-large wp-image-1229 alignnone" src="http://scienceblogs.com/digitalbio/files/2016/09/CRISPR_tutorial_2-400x324.png" alt="crispr_tutorial_2" width="400" height="324" /></p> <p style="padding-left: 30px;">6.  Select the DNA and RNA and apply the residue coloring style.</p> <p style="padding-left: 30px;"><img class="size-large wp-image-1230 alignnone" src="http://scienceblogs.com/digitalbio/files/2016/09/CRISPR_tutorial_3-400x369.png" alt="crispr_tutorial_3" width="400" height="369" /></p> <p style="padding-left: 30px;">7.  Deselect the DNA, hide the unselected residues, and hide the sequence viewer.</p> <p style="padding-left: 30px;"><img class="size-large wp-image-1231 alignnone" src="http://scienceblogs.com/digitalbio/files/2016/09/CRISPR_tutorial_4-400x348.png" alt="crispr_tutorial_4" width="400" height="348" /></p> <p style="padding-left: 30px;">8.  Turn the structure around to view it from all angles.</p> <p style="padding-left: 60px;">Q1.  Do you see anything interesting about the structure of the RNA?</p> <p style="padding-left: 60px;">This is the guide RNA.  It has a special shape that's recognized by the Cas protein.</p> <p style="padding-left: 30px;">9.  Select the DNA and hide everything else.</p> <p style="padding-left: 60px;">Q2.  Where do you think the DNA gets cut?</p> <p style="padding-left: 30px;">10.  Lock the DNA and protein chains.</p> <p style="padding-left: 30px;"><img class="size-large wp-image-1232 alignnone" src="http://scienceblogs.com/digitalbio/files/2016/09/CRISPR_tutorial_5-400x339.png" alt="crispr_tutorial_5" width="400" height="339" /></p> <p style="padding-left: 30px;">11.  Select nearby residues to see how part of the RNA interacts with the target DNA.</p> <p style="padding-left: 30px;"><img class="size-large wp-image-1233 alignnone" src="http://scienceblogs.com/digitalbio/files/2016/09/CRISPR_tutorial_6-400x313.png" alt="crispr_tutorial_6" width="400" height="313" /></p> <p style="padding-left: 60px;">Q3.  How does the selected part of the RNA interact with the target DNA?</p> <p style="padding-left: 30px;">12.  Hide the unselected residues.</p> <p style="padding-left: 60px;">Q4.  Now, where do you think the DNA gets cut?</p> <p style="padding-left: 30px;">13.  Look at the RNA sequence.  Select the base on the 3' side of the last highlighted base.  (This should be a U).</p> <p style="padding-left: 30px;"><img class="size-large wp-image-1234 alignnone" src="http://scienceblogs.com/digitalbio/files/2016/09/CRISPR_tutorial_7-400x316.png" alt="crispr_tutorial_7" width="400" height="316" /></p> <p style="padding-left: 30px;">14.  Open the color key.</p> <p style="padding-left: 60px;">Q5.  What kinds of bases are forming pairs?  Notice the RNA and DNA sequences are complementary.</p> <p style="padding-left: 90px;">The GUU sequence is a special motif that helps control where the DNA gets cut.</p> <p style="padding-left: 60px;">Q6.  What part of the RNA sequence would you change if you wanted to cut a different sequence of DNA?</p> <p style="padding-left: 30px;">Hint:  <em>It may help, to unlock the chains and apply Rainbow coloring to identify the 5' and 3' ends of the DNA and RNA molecules.</em></p> </div> <span><a title="View user profile." href="/author/sporte">sporte</a></span> <span>Sun, 09/18/2016 - 12:51</span> Sun, 18 Sep 2016 16:51:20 +0000 sporte 69984 at https://scienceblogs.com Zika virus, drug discovery, and student projects https://scienceblogs.com/digitalbio/2016/03/09/zika-virus-drug-discovery-and-student-projects <span>Zika virus, drug discovery, and student projects</span> <div class="field field--name-body field--type-text-with-summary field--label-hidden field--item"><p>It's well understood in science education that students are more engaged when they work on problems that matter.  Right now, Zika virus matters.  Zika is a very scary problem that matters a great deal to anyone who might want to start a family and greatly concerns my students.</p> <p>I teach a bioinformatics course where students use computational tools to research biology.  Since my students are learning how to use tools that can be applied to this problem, I decided to have them apply their new bioinformatics skills to identify drugs that work against Zika virus.</p> <p>We don't have the lab facilities to test drug candidates, but it's nice for students to realize they're learning skills that could be put to use.</p> <p>Here's what we're doing:</p> <ol><li>Looking at background information about Zika virus.</li> <li>Using blastp to identify related proteins that are also bound to drugs.</li> <li>Using molecular modeling to see if those drugs might also bind to Zika virus proteins.</li> </ol><p> </p> <p><strong>Getting up-to-speed on Zika virus</strong></p> <p>We found a great compilation of <a href="http://www.ncbi.nlm.nih.gov/genome/viruses/variation/Zika/" target="_blank">Zika resources at the NCBI</a>.  <a href="http://www.cidrap.umn.edu/infectious-disease-topics/zika#literature" target="_blank">CIDRAP has a great set of Zika resources</a> as well.</p> <p>My students go to the NCBI Zika resource, select the link to publications, and scan the titles to see what's new.  This list is a bit overwhelming, so I ask them to focus on the first and last sentences in the abstract from P. Brasil et. al., <a href="http://www.ncbi.nlm.nih.gov/pubmed/26943629" target="_blank">Zika Virus Infection in Pregnant Women in Rio de Janeiro</a>, and on <a href="http://www.sciencedirect.com/science/article/pii/S1934590916001065" target="_blank">this publication from Tang, et. al</a>.  They need to identify birth defects associated with Zika virus infection and summarize two kinds of data that support the association between infection and birth defects.</p> <p>Next, they use the <a href="http://www.healthmap.org/zika/#timeline" target="_blank">Health Map</a> link to see where infections are occurring.  It gets more personal when you see cases happening in your state.</p> <div style="width: 410px;float:left;"><img class="size-large wp-image-1214" src="http://scienceblogs.com/digitalbio/files/2016/03/Healthmap-400x239.png" alt="Health Map shows Zika virus cases in real time." width="400" height="239" /><a href="http://www.healthmap.org/zika/#timeline" target="_blank">Health Map </a>shows Zika virus cases in real time. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>We also look at the <a href="http://viralzone.expasy.org/all_by_species/6756.html" target="_blank">ViralZone page from Expasy</a> to learn about the Zika life cycle and see how the Zika polyprotein gets chopped into smaller parts.  This has a link to an interesting Wikipedia page for a Zika virus receptor (<a href="https://en.wikipedia.org/wiki/DC-SIGN" target="_blank">DC-SIGN or CD209</a>) that appears to be expressed in the uterus and on brain cells–at least that's my interpretation of the RNA expression data.</p> <p>But, it's easy to get lost clicking too many links, so we go on to protein blast.</p> <p> </p> <p><strong>Identifying potential drug targets with BLAST</strong></p> <p>I think the easiest way to find a drug against a virus is to start by looking at compounds we already know about.  We know that many successful antiviral drugs target viral proteases and polymerases, so my students go to the Zika virus reference genome (thanks NCBI!) and get the protein sequences for the <a href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Search&amp;db=protein&amp;term=YP_009227202.1&amp;dopt=GenBank" target="_blank">Zika virus protease NS3</a> and the <a href="http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Search&amp;db=protein&amp;term=YP_009227205.1&amp;dopt=GenBank" target="_blank">Zika virus RNA dependent RNA polymerase</a>.</p> <p>Then they use protein blast to search the NCBI structure database and see if there are 3D structures from related viruses that are bound to drugs.</p> <p>Once they've found a structure to work with, they reverse the search and use blastp to compare their new sequence to the sequence of the Zika protein.</p> <p> </p> <p><strong>Using molecular models to see if drugs might bind to Zika virus</strong></p> <p>Once our students have found structures that contain a drug, they look at amino acids that are near the drug to see if those residues are similar to those in Zika virus.</p> <p> </p> <p><strong>Would Sovaldi® (Sofosbuvir) work against Zika virus?</strong></p> <p>Whenever possible, I like to give examples to show an investigation might work.  When I noticed that some of my blast results included proteins from Hepatitis C virus, I decided to use this as an example.  There's a drug that works by inhibiting the RNA polymerase in Hepatitis C  (<a href="http://www.sovaldi.com" target="_blank">Sovaldi® from Gilead</a>), so I decided to find out if it might work against Zika as well.</p> <div style="width: 410px;float:left;"><img class="size-large wp-image-1216" src="http://scienceblogs.com/digitalbio/files/2016/03/IMG_2927-400x283.png" alt="Hepatitis C virus RNA polymerase bound to Sovadi® (Sofosbuvir) from 4WTG colored by charge." width="400" height="283" /> Hepatitis C virus RNA polymerase bound to Sovadi® (Sofosbuvir) from 4WTG colored by charge. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>First, I did a blastp search and compared the protein sequence from the structure 4WTG against Zika virus RNA polymerase.</p> <div style="width: 410px;float:left;"><img class="size-large wp-image-1215" src="http://scienceblogs.com/digitalbio/files/2016/03/HepC_vZika-400x101.png" alt="blastp results from comparing Zika virus RNA polymerase to the Hepatitis C virus polymerase in 4WTG" width="400" height="101" /> blastp results from comparing Zika virus RNA polymerase to the Hepatitis C virus polymerase in 4WTG. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>Only 25% of the amino acids are identical, but the E value is 0.007, so that's encouraging.   I decided to take a closer look.</p> <p>I used 4WTG as a query sequence in blastp to align it to the Zika virus polymerase sequence.  Then, I downloaded the 4WTG structure and opened it in Molecule World. I selected the drug and used the Select Nearby feature to identify amino acids that might be bound to the drug. Returning to the aligned sequences, I highlighted those amino acids in the alignment.</p> <p><img class="alignleft size-large wp-image-1213" src="http://scienceblogs.com/digitalbio/files/2016/03/Blastp_HepC_Zika-400x85.png" alt="Blastp_HepC_Zika" width="400" height="85" /></p> <p> </p> <p> </p> <p> </p> <p>Interestingly, the drug binds to amino acids that are present in the same positions in both Zika virus RNA polymerase and in the Hepatitis C virus RNA polymerase.  Cool!</p> <p>I took a closer look.  In the top image, two manganese atoms bound to the drug are also bound to aspartic acid residues.  These are present in both proteins.</p> <div style="width: 410px;float:left;"><img class="size-large wp-image-1211" src="http://scienceblogs.com/digitalbio/files/2016/03/IMG_2924-400x283.png" alt="Amino acids that interact with Sovaldi® are colored by residue in Molecule World and drawn as tubes." width="400" height="283" /> Amino acids that interact with Sovaldi® are colored by residue in Molecule World and drawn as tubes. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>In the bottom image, I can see an arginine that's present in both proteins.  Here, it appears to participate in an ionic interaction with the drug.</p> <div style="width: 410px;float:left;"><img class="size-large wp-image-1212" src="http://scienceblogs.com/digitalbio/files/2016/03/IMG_2925-400x283.png" alt="Amino acids that interact with Sovaldi® are drawn with in a space filling mode and colored by element in Molecule World." width="400" height="283" /> Amino acids that interact with Sovaldi® are drawn with in a space filling mode and colored by element in Molecule World. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>Now, these models don't prove that Sovaldi would inhibit Zika virus replication.  But it might be worth taking a look.  If I were culturing brain stem cells like Tang, et. al (3), I might take out a loan to buy some Sovaldi® and add it to the growth medium.   Just to see what happens.</p> <p>For now, I'm looking forward to seeing what my students find.</p> <p>Note:  All the molecular modeling work described here was carried out with the <a href="https://geo.itunes.apple.com/us/app/molecule-world/id863565223?mt=8&amp;at=10lGBR&amp;ct=zika" target="_blank">Molecule World iPad app</a> from Digital World Biology.</p> <p> </p> <p><strong>References</strong>:</p> <ol><li> <a href="http://www.ncbi.nlm.nih.gov/genome/viruses/variation/Zika/">The Zika Virus Resource at the National Center for Biotechnology Information</a></li> <li> <p class="desc">Brasil P, et.al.  <a href="http://www.ncbi.nlm.nih.gov/pubmed/26943629" target="_blank">Zika Virus Infection in Pregnant Women in Rio de Janeiro</a> <span class="jrnl" title="The New England journal of medicine">N Engl J Med</span>. 2016 Mar 4. [Epub ahead of print]</p> </li> <li>Tang et al., Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth, Cell Stem Cell (2016),  <a href="http://dx.doi.org/10.1016/j.stem.2016.02.016">http://dx.doi.org/10.1016/j.stem.2016.02.016</a></li> <li>Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.</li> <li>Appleby TC, Perry JK, Murakami E, Barauskas O, Feng J, Cho A, Fox D 3rd,<br /> Wetmore DR, McGrath ME, Ray AS, Sofia MJ, Swaminathan S, Edwards TE. <a href="http://www.ncbi.nlm.nih.gov/pubmed/25678663">Viral</a><br /><a href="http://www.ncbi.nlm.nih.gov/pubmed/25678663"> replication. Structural basis for RNA replication by the hepatitis C virus</a><br /><a href="http://www.ncbi.nlm.nih.gov/pubmed/25678663"> polymerase</a>. Science. 2015 Feb 13;347(6223):771-5. doi: 10.1126/science.1259210.</li> </ol><p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> </div> <span><a title="View user profile." href="/author/sporte">sporte</a></span> <span>Tue, 03/08/2016 - 18:10</span> Tue, 08 Mar 2016 23:10:56 +0000 sporte 69982 at https://scienceblogs.com DNA: it's in your blood https://scienceblogs.com/digitalbio/2016/02/28/dna-its-in-your-blood <span>DNA: it&#039;s in your blood</span> <div class="field field--name-body field--type-text-with-summary field--label-hidden field--item"><p>Did you know small fragments of DNA are circulating in your blood stream?</p> <p>These short pieces of DNA are left behind after cells self-destruct. This self-destruction, or apoptosis, is a normal process. In the case of fetal development, certain cells in our hands die, leaving behind individual fingers. Immune system cells leave traces of DNA behind after they’ve tackled invading microbes. DNA can also appear in the blood when people have cancer.</p> <p>I had the good fortune, last Monday, to hear Matthew Snyder describe this cell-free DNA in a fascinating talk and learn why DNA in the blood can be a useful thing. It turns out that cell free DNA is a potentially useful tool for evaluating fetal health, guiding cancer treatment, and monitoring organ transplants.</p> <p>According to Snyder, the use of cell free DNA for diagnosing trisomy 21, is one of the fastest growing molecular tests in the history of medicine.  Some of the rapid adoption of this test is driven by pregnant women who request it.</p> <p>People are interested in the prospect of using cell free DNA for other kinds of tests as well. It could be used as a biomarker to indicate the presence of cancer, or perhaps other kinds of disease.</p> <p>Snyder’s research involves sequencing this cell free DNA and trying to figure out where it came from. You might think that the DNA in one person would be pretty much the same from one cell to another. And with a few exceptions, like B and T cells, that’s the case. But the DNA fragments that float around in our blood aren’t random. We can only find DNA fragments in our blood because they were hidden from hungry nucleases during the self-destruction process. Normally, those enzymes would have chopped that DNA into tiny bits.</p> <p>Cell free DNA exists because the proteins that transcribe DNA and the histone proteins that package it into nucleosomes also protect DNA from being digested.</p> <div style="width: 460px;float:left;"><img class="size-full wp-image-700" src="/files/digitalbio/files/2012/04/8647-Digital_genomic_footprinting.gif" alt="Proteins protecting DNA from digestion." width="450" height="625" /> Proteins protecting DNA from digestion. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>The really interesting thing, in terms of cell free DNA, is that nucleosomes and transcription factors sit on different regions of DNA in different cells.  Since different bits of DNA get protected in different cells, we can sequence the cell free bits of DNA figure out where it came from.  That information can tell us about a type of cancer or help us evaluate the health of multiple cell types.</p> <div style="width: 410px;float:left;"><img class="size-large wp-image-1208" src="http://scienceblogs.com/digitalbio/files/2016/02/nucleosome-400x373.png" alt="This structure has been colored by charge. The negatively charged DNA (red) is wrapped about the positively charged histone proteins. Blue represents a positive charge." width="400" height="373" /> This structure has been colored by charge. The negatively charged DNA (red) is wrapped about the positively charged histone proteins. Blue represents a positive charge. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p><strong>Reference</strong>:</p> <p>Snyder MW, Kircher M, Hill AJ, Daza RM, Shendure J. <a href="http://www.ncbi.nlm.nih.gov/pubmed/26771485">Cell-free DNA Comprises an In Vivo Nucleosome Footprint that Informs Its Tissues-Of-Origin</a>. Cell. 2016 Jan 14;164(1-2):57-68. doi: 10.1016/j.cell.2015.11.050.</p> </div> <span><a title="View user profile." href="/author/sporte">sporte</a></span> <span>Sun, 02/28/2016 - 14:20</span> Sun, 28 Feb 2016 19:20:22 +0000 sporte 69981 at https://scienceblogs.com Protein modeling and the Siamese cat https://scienceblogs.com/digitalbio/2015/12/31/protein-modeling-and-the-siamese-cat <span>Protein modeling and the Siamese cat</span> <div class="field field--name-body field--type-text-with-summary field--label-hidden field--item"><p>"<em>By night all cats are gray</em>"  - Miguel Cervantes in Don Quixote</p> <p> </p> <p>I've always liked Siamese cats.   Students do, too.  "Why Siamese cats wear masks" is always a favorite story in genetics class.  So, when I opened my January copy of <a href="http://www.nsta.org/highschool/" target="_blank">The Science Teacher</a>, I was thrilled to see an article on Siamese cat colors and proteins AND molecular genetics (1).</p> <p>In the article, the authors (Todd and Kenyon) provide some background information on the enzymatic activity of tyrosinase and compare it to the catechol oxidase that causes fruit to brown, especially apples.  Tyrosinase catalyzes the first step of a biochemical pathway where tyrosine is converted to dopaquinone and then to the dark colored substance, melanin.  Melanin is responsible for the brown color in the ears, paws, tail, and face of Siamese cats.</p> <div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2016/01/melanin.png"><img class="size-large wp-image-1195" src="http://scienceblogs.com/digitalbio/files/2016/01/melanin-400x320.png" alt="Melanin in Molecule World" width="400" height="320" /></a> Melanin in Molecule World </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>The fun part of the story is that some versions of the tyrosinase gene have a mutation that makes this enzyme less stable at warmer temperatures.  In warmer areas of the cat's body, less melanin is produced, giving the cat a lighter colored body.</p> <p>I love the story of the coat coloring  and I think this activity has the potential to interest students.</p> <p>I also like the approach that the authors take with discussing proteins and the associated phenotypes first before getting into DNA and the central dogma.  I use this same order in my bioinformatics class.</p> <p>There are, however, a few things that I would change with this activity.</p> <p><strong>Too many hydroxyl groups</strong></p> <p>First, the article and the teacher guide have the structure of tyrosine wrong.  Tyrosine only has one hydroxyl group attached to the six-carbon ring, as shown below, not two hydroxyl groups as shown in the article and the teachers' guide.</p> <div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2016/01/extra-tyrosine.png"><img class="size-large wp-image-1196" src="http://scienceblogs.com/digitalbio/files/2016/01/extra-tyrosine-400x155.png" alt="Tyrosinase reaction from Teachers' Guide. The extra hydroxyl group is marked." width="400" height="155" /></a> Tyrosinase reaction from Teachers' Guide. The extra hydroxyl group is marked. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>Space-filling models of Tyrosine and Dopaquinone downloaded from the NCBI's PubChem database are shown below.</p> <div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/12/tyrosine_dopaquinone.png"><img class="size-large wp-image-1191" src="http://scienceblogs.com/digitalbio/files/2015/12/tyrosine_dopaquinone-400x186.png" alt="Tyrosine and dopaquinone" width="400" height="186" /></a> Tyrosine and dopaquinone </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p><strong>Too many proteins in too short a time</strong></p> <p>Second, I think the authors include way too many different protein stories and different explanations for why a protein might be inactivated.  Todd and Kenyon have done quite a bit of work creating activities with several different proteins. But I think the plan to have students cover all eight proteins in six days would be too confusing for many students.  The activity has students read about or work with:  lactase, cellulase, tyrosinase, catechol oxidase, proteases, kinesin, galactosyl transferase, and tyrosine kinase, not to mention Gleevec and the way it interacts with the BCR-ABL fusion protein.  I teach a college bioinformatics course and even the best of my students would get confused by looking at eight different proteins without some kind of common thread.  The take home lesson that denaturation negatively impacts tyrosinase activity is easy to lose among all the unrelated activities.</p> <p><strong>Where are the 3D models?</strong></p> <p>When I read the description of the article, I thought it might include activities where students looked at 3D protein models to see how they changed when they were denatured by heat or pH. Instead, they just watch a short video. A better approach, I think, would be to have students work first-hand with structure models and see the change for themselves.  Or maybe fry and egg.  The effects of protein denaturation are pretty clear when you watch an egg cook.</p> <p>This is my bias, but I think an active learning approach, where students actually look at 3D protein and chemical models and identify chemical interactions, would be better in long run and better equip students for future learning. I think the common practice of hiding the biochemistry makes genetics much harder to understand and far less straightforward than it should be.</p> <p>And, as it turns out there are 3D models of tyrosinase that students could use.  They're from <em>Bacillus megatherium</em>, but that's how biology works.  If an enzyme activity is beneficial, evolutionary processes tend to keep it around.</p> <p><strong>Using 3D Models to look at albino cats</strong></p> <p>No models exist from the Siamese cat protein with the brown ear point mutation, but it is possible to make 3D protein models that show the affect of a different mutation, in tyrosinase, that leads to albino cats. This mutation occurs when a cytosine is deleted at nucleotide 975, creating a frameshift (2).  To simulate the mutation's effect, we can hide the amino acids that would be lost.  This model isn't perfect because the shorter protein might fold somewhat differently, but this does provide a satisfying explanation for an inactive enzyme.</p> <p>Here's what to do:</p> <ol><li>Open <a href="https://geo.itunes.apple.com/us/app/molecule-world/id863565223?mt=8&amp;at=10lGBR&amp;ct=cat" target="_blank">Molecule World on the iPad</a> (*).</li> <li>Download <a href="http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdb_strview.cgi/mmdb.cn3?program=cn3d&amp;display=0&amp;complexity=3&amp;buidx=1&amp;uid=121933" target="_blank">4P6R</a>.</li> <li>Color the protein chains by molecule.</li> <li>Open the sequence viewer and touch the name of the last row to select it.  This row contains tyrosine, the substrate for the enzyme, and for each chain, two atoms of zinc.</li> <li>Change the coloring style to element.</li> </ol><p>Now, you can see the substrate bound in the active site.  Notice this protein has two identical subunits.  Each one is bound to tyrosine and zinc.</p> <div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/12/tyrosinase_normal.png"><img class="size-large wp-image-1192" src="http://scienceblogs.com/digitalbio/files/2015/12/tyrosinase_normal-400x303.png" alt="Tyrosinase with tyrosine and zinc in the active site." width="400" height="303" /></a> Tyrosinase with tyrosine and zinc in the active site. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>You may want to change the atom visibility to show all the atoms or just the core backbone, like I have here, to see how the tyrosine is positioned.</p> <p>*Many of the things I describe here can be done in Cn3D, but it's a bit more complicated.</p> <p> </p> <p><strong>Modeling the mutation's affect</strong></p> <p>This next part is a little more complicated because I had to split the protein sequence into three parts to avoid introducing spaces.</p> <ol><ol><li>Carry out the following process in three steps. First, copy the sequence below.KYRVRKNVLHLTDTEKRDFVRTVLIKEKGIYDRYIAWHGAAGKFHTPPGSDRNA</li> </ol></ol><p> </p> <ol><ol><li>Touch the Selection button and paste the sequence below in the Select pattern window.</li> <li>Start the search.  The pasted sequence will be highlighted in the protein sequence.</li> <li>Copy the next part of the sequence (below).  Touch the Selection button again and search for this pattern as before.<br /> AHMSSAFLPWHREYLLRFERDQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGN</li> </ol></ol><p> </p> <ol><ol><li>Copy the next part of the sequence (below).  Touch the Selection button again and search for this pattern as before.<br /> GNPIKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKAPTLPTRDDVL</li> </ol></ol><p> </p> <ol><li>When you're done highlighting sequences, you'll see each subunit has a region that appears brighter and a region in the center that appears more dim. The bright colored residues are incorporated into the protein before ribosomes encounter the frame shift mutation and a stop codon shortly afterwards. This portion of the protein can be produced in the albino cat.</li> <li>Look at the dim areas of each subunit.  These amino acids would be lost when the frameshift mutation is present.  Notice where the tyrosine is located.</li> </ol><div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/12/tyrosinase_affected.png"><img class="size-large wp-image-1193" src="http://scienceblogs.com/digitalbio/files/2015/12/tyrosinase_affected-400x326.png" alt="Residues that would be lost because of the frameshift mutant are shown in gray." width="400" height="326" /></a> Residues that would be lost because of the frameshift mutant are shown in gray. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>8.  To make the mutations affect on the protein even more clear, open the Show/Hide button and choose "Hide unselected."  The amino acid residues that would be lost because of the mutation disappear.</p> <div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/12/tyrosinase_missing.png"><img class="size-large wp-image-1194" src="http://scienceblogs.com/digitalbio/files/2015/12/tyrosinase_missing-400x285.png" alt="Residues that would be lost because of the frame shift mutation are hidden." width="400" height="285" /></a> Residues that would be lost because of the frame shift mutation are hidden. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>Now, it's really clear.  If the residues that bind tyrosine and modify it's structure are gone, the enzyme is unable to function.  If we don't have tyrosinase working to help make melanin, we get white cats.</p> <p> </p> <p><strong>References</strong>:</p> <ol><li>Amber Todd and Lisa Kenyon, How do Siamese cats get their color? The Science Teacher. 2016;83(1):29-36</li> <li>Imes D, Geary L, Grahn R, Lyons L. Albinism in the domestic cat (<i>Felis catus</i>) is associated with a <i>tyrosinase</i> (<i>TYR</i>) mutation. <i>Animal Genetics</i>. 2006;37(2):175-178. doi:10.1111/j.1365-2052.2005.01409.x.</li> </ol></div> <span><a title="View user profile." href="/author/sporte">sporte</a></span> <span>Thu, 12/31/2015 - 01:00</span> Thu, 31 Dec 2015 06:00:03 +0000 sporte 69979 at https://scienceblogs.com Exploring levels of protein structure with molecular models and snake venom https://scienceblogs.com/digitalbio/2015/12/18/exploring-levels-of-protein-structure-with-molecular-models-and-snake-venom <span>Exploring levels of protein structure with molecular models and snake venom</span> <div class="field field--name-body field--type-text-with-summary field--label-hidden field--item"><p>Imagine a simple hike in a grassy part of South America.  You hear a rattle and feel a quick stab of pain as fangs sink into your leg.  Toxins in the snake venom travel through your blood vessels and penetrate your skin.  If the snake is a South American rattlesnake, <em>Crotalus terrific duressis</em>, one of those toxins will be a phospholipase.  Phospholipases attack cell and mitochondrial membranes destroying nerve and muscle function.  Without quick treatment, a snakebite victim may be die or suffer permanent damage (1, 2).</p> <p>The phospholipase from the South American rattlesnake is called crotoxin.  Scientists are interested in studying crotoxin because snake bites are a serious health hazard and better treatments would help save lives and minimize nerve and muscle damage.</p> <p>Today, we're going to explore crotoxin for a different reason.  We're going to use crotoxin to investigate the four levels of protein structure. Crotoxin is a good protein for this activity because it contains both types of secondary structure, metals, disulfide bonds, and multiple protein chains.  Some proteins don't work as well for this purpose because they are monomeric, or they only contain one type of secondary structure.</p> <p>We'll use <strong><a href="https://geo.itunes.apple.com/us/app/molecule-world/id863565223?mt=8&amp;at=10lGBR&amp;ct=levels" target="_blank">Molecule World</a> </strong>for this activity, but other 3D modeling programs could also be used.  Some people also use plastic tubes and magnets but if you have access to iPads, it makes sense to use actual structure models.</p> <p>The structure we're going to use is <a href="http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdb_strview.cgi/mmdb.cn3?program=cn3d&amp;display=0&amp;complexity=3&amp;buidx=1&amp;uid=94243" target="_blank">3R0L</a>.  If you touch the link on an iPad, the structure will be downloaded from the NCBI and you can open it in Molecule World.</p> <p><strong>Primary structure</strong></p> <p>The primary structure of a protein is the sequence of amino acids, joined in a chain, from the amino end to the carboxyl end.  Crotoxin has four protein chains, each with a different sequence.</p> <p>To view the amino acid sequences:</p> <ol><li>Open the sequence viewer.</li> <li>Touch the atom icon and choose residues to see how the sequences in four chains are different.</li> <li>If you open the color key, you can see the colors and abbreviations for each amino acid.</li> </ol><div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/12/IMG_2542.png"><img class="wp-image-1180 size-large" src="http://scienceblogs.com/digitalbio/files/2015/12/IMG_2542-400x274.png" alt="IMG_2542" width="400" height="274" /></a> When residue coloring is used, each amino acid appears with a different color. We can see that these four amino acid sequences are all different. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p><strong>Secondary structure</strong></p> <p>The two kinds of secondary structure elements are alpha helices and beta sheets.  These distinctive shapes are held together by interactions between atoms in the amino acid backbone.</p> <p>To view both types of secondary structure:</p> <ol><li>Touch the atom icon and choose Secondary view.</li> <li>Use the color key to identify both types in the structure.</li> </ol><div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/12/IMG_2543.png"><img class="wp-image-1181 size-large" src="http://scienceblogs.com/digitalbio/files/2015/12/IMG_2543-400x292.png" alt="IMG_2543" width="400" height="292" /></a> Both types of secondary structure are shown in this image of the crotoxin B subunit. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p><strong>A.  Explore an alpha helix</strong></p> <p>To see how an alpha helix is held together:</p> <ol><li> Find chain 3R0L-D in the sequence viewer.</li> <li>Touch the first set of green letters (LLQFNKMIKFE).  One alpha helix will appear brighter.</li> <li>Open the Show/Hide menu and choose Hide unselected. The rest of the structure will dissappear.</li> </ol><p><a href="/files/digitalbio/files/2015/12/IMG_2544.png"><img class="alignleft size-large wp-image-1182" src="http://scienceblogs.com/digitalbio/files/2015/12/IMG_2544-400x300.png" alt="IMG_2544" width="400" height="300" /></a></p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <ol><li>Touch the atom icon and pick the element coloring style.</li> <li>Open the Show/Hide menu and choose Complete backbone.</li> <li>Use the color key to identify the different atoms.</li> <li>Open the Show/Hide menu again and choose All atoms in residue.  Now you can see where the side chains are positioned.</li> </ol><div style="width: 321px;float:left;"><a href="/files/digitalbio/files/2015/12/IMG_2557.png"><img class="wp-image-1183 size-full" src="/files/digitalbio/files/2015/12/IMG_2557.png" alt="IMG_2557" width="311" height="230" /></a> From left to right, this alpha helix shows the amino acid backbone, the backbone plus the oxygens, and the backbone plus the side chains. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>In an alpha helix, hydrogen bonds (invisible in this structure) form between the oxygens (red) and nitrogens (blue) in the backbone and hold the structure in a helical shape.</p> <p>Touch the atom icon and Choose Reset View before going on to the beta sheets.</p> <p> </p> <p><strong>B.  Explore beta sheets</strong></p> <p>To see how beta sheets are held together:</p> <ol><li>Touch the atom icon and choose Secondary view.</li> <li>Scroll through the sequence viewer to find where the beta sheet begins.</li> <li>Touch all the letters in the beta sheet.</li> <li>Open the Show/Hide menu and choose Hide unselected.</li> <li>Touch the atom icon and pick element coloring style.  The beta sheet looks like a loop.</li> <li>Open the Show/Hide menu and choose Complete backbone. This time, you can see the interactions between oxygens and nitrogens in different loops of the chain.</li> <li>Open the Show/Hide menu again and choose All atoms in residue to see where the side chains are located.</li> </ol><div style="width: 287px;float:left;"><a href="/files/digitalbio/files/2015/12/IMG_2558.png"><img class="wp-image-1184 size-full" src="/files/digitalbio/files/2015/12/IMG_2558.png" alt="IMG_2558" width="277" height="172" /></a> From left to right, this beta sheet shows the amino acid backbone only, the backbone with oxygens, and the backbone plus the side chains. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p><strong>Tertiary structure</strong></p> <p>The tertiary structure of a protein consists of a single amino acid chain plus any metals or prosthetic groups like heme or NAD.</p> <p>Touch the atom icon and Choose Reset View before going on.</p> <p>To see an example of tertiary structure:</p> <ol><li>Touch the names 3R0L-D and 3R0L-other.  The "other" row is a list of chemicals in the structure.</li> <li>Open the Show/Hide menu and choose Hide unselected.  The objects that remain are a tertiary structure.</li> <li>Touch the atom icon and choose Secondary view.  You can see this chain contains both types of secondary structure.</li> </ol><p> </p> <p><strong>Explore bonds to metals</strong></p> <p>Touch the atom icon and Choose Reset View before going on.</p> <ol><li>Touch the name 3R0L-D.</li> <li>Open the Show-Hide menu and choose Hide unselected.</li> <li>Touch the name 3R0L-D again to deselect it.</li> <li>Touch the Mn (manganese).</li> <li>Open the Selection menu and choose Select nearby.</li> <li>Open the Show/Hide menu and choose All atoms in residue.</li> <li>Touch the atom icon and choose the Ball and Stick drawing style and element coloring style.  Now, you can see there are bonds holding the manganese atom in place.  Similar bonds hold the sodium atoms in place as well.</li> </ol><div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/12/IMG_2554.png"><img class="wp-image-1185 size-large" src="http://scienceblogs.com/digitalbio/files/2015/12/IMG_2554-400x283.png" alt="IMG_2554" width="400" height="283" /></a> Oxygens on amino acid side chains form bonds to manganese and hold it in place. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p><strong>Explore disulfide bonds</strong></p> <p>Touch the atom icon and Choose Reset View before going on.</p> <ol><li>Touch the atom icon and choose Secondary View.</li> <li>Open the Selection menu and type C.</li> <li>Open the Show/Hide menu and choose All atoms in residue.</li> <li>Touch the atom icon and select the Ball and stick drawing style and element coloring style.</li> <li>Touch the names of chains A, B, and C twice to select and deselect them.  Now, the cysteines will only be selected in chain D.</li> <li>Touch 3R0L-D to select chain D.</li> <li>Open the Show/Hide menu and choose Hide unselected.  The disulfide bonds between chains can be seen.</li> </ol><div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/12/IMG_2545.png"><img class="wp-image-1186 size-large" src="http://scienceblogs.com/digitalbio/files/2015/12/IMG_2545-400x354.png" alt="IMG_2545" width="400" height="354" /></a> Yellow disulfide bonds hold alpha helices together in crotoxin. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p><strong>Quaternary structure</strong></p> <p>The quaternary structure of a protein consists of the interactions between multiple chains.</p> <p>Touch the atom icon and Choose Reset View before going on.</p> <p>To view the quaternary structure of this enzyme:</p> <ol><li> Touch the atom icon and the Spacefill drawing style and molecule coloring style.</li> <li>Open the Show/Hide menu and choose all atoms in residue.  You can see all four chains.</li> </ol><p> </p> <p>One last fun thing we can do with this this protein is to identify the active site.  In crotoxin, the active site is the place where this enzyme chops up phospholipids.</p> <p><strong>Exploring the active site</strong></p> <p>To explore the active site:</p> <ol><li> Touch the atom icon and Choose Reset View before going on.</li> <li>Touch the name of chain 3R0L-D to select it.</li> <li>Open the Show/Hide menu and choose Hide unselected to hide chains A, B, and C.  These chains are involved in binding to the cell membrane but they don't contribute to the phospholipase activity.</li> <li>Touch the atom icon and choose the hydrophobicity coloring style and the spacefill drawing style.</li> <li>Open the Show/Hide menu and choose Hide unselected.</li> <li>Open the Show/Hide menu again and choose All atoms in residue.</li> <li>Touch ACT in the 3R0L-other chain to highlight acetate.  Acetate is a product of phospholipase activity.</li> <li>Hide the sequence viewer and turn the structure around to view the acetate inside the enzyme at the active site.</li> </ol><div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/12/IMG_2556.png"><img class="size-large wp-image-1187" src="http://scienceblogs.com/digitalbio/files/2015/12/IMG_2556-400x287.png" alt="Crotoxin colored by hydrophobicity with acetate bound in the active site." width="400" height="287" /></a> Crotoxin colored by hydrophobicity with acetate bound in the active site. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p><strong>References</strong></p> <ol><li>Harris JB, Scott-Davey T. Secreted phospholipases A2 of snake venoms: effects on the peripheral neuromuscular system with comments on the role of phospholipases A2 in disorders of the CNS and their uses in industry. Toxins (Basel). 2013 Dec 17;5(12):2533-71. doi: 10.3390/toxins5122533.</li> <li>Faure G, Xu H, Saul FA. Crystal structure of crotoxin reveals key residues involved in the stability and toxicity of this potent heterodimeric β-neurotoxin. J Mol Biol. 2011 Sep 16;412(2):176-91. doi: 10.1016/j.jmb.2011.07.027.</li> </ol></div> <span><a title="View user profile." href="/author/sporte">sporte</a></span> <span>Thu, 12/17/2015 - 18:47</span> Thu, 17 Dec 2015 23:47:52 +0000 sporte 69978 at https://scienceblogs.com The invisible data of Isabella Karle https://scienceblogs.com/digitalbio/2015/12/01/the-invisible-data-of-isabella-karle <span>The invisible data of Isabella Karle</span> <div class="field field--name-body field--type-text-with-summary field--label-hidden field--item"><p>When finding a female scientists' data turns into an archeological treasure hunt.</p> <p>A few months ago, I decided it would be interesting to celebrate various scientific contributions by making images of chemical / molecular structures in the <a href="https://geo.itunes.apple.com/us/app/molecule-world/id863565223?mt=8&amp;at=10lGBR&amp;ct=blog" target="_blank">Molecule World iPad app</a> and posting them on Twitter  (<a href="http://scienceblogs.com/digitalbio/2015/09/08/making-the-most-of-molecule-of-the-month-with-molecule-world/" target="_blank">@MoleculeWorld</a>).  Whenever I can, I like to highlight scientific contributions from women on their birthdays.  Tomorrow's post will feature Dr. Isabella Karle, an x-ray crystallographer who worked on the Manhattan project and solved structures of interesting molecules like valinomycin and a South American frog venom (1).</p> <div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/12/IMG_2493.png"><img class="size-large wp-image-1176" src="http://scienceblogs.com/digitalbio/files/2015/12/IMG_2493-400x283.png" alt="Valinomycin in Molecule World." width="400" height="283" /></a> Valinomycin in Molecule World. The structure was obtained from ChemSpider (2) and converted to a PDB file for viewing. This antibiotic makes a channel in the plasma membrane causing potassium to leak out and triggering apoptosis (3) </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>Reading about Dr. Karle's many accomplishments in Wikipedia made me think it should be easy to find and view some of the structures she solved. Indeed, I found <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Karle%2C+Isabella" target="_blank">18 papers in PubMed</a>, where she's listed as an author. Most impressively, if this is the same IL Karle, one paper dates from 2012, making her almost 91 at the time of publication!</p> <p>But in the quest for structures, the results were nil.  Searching the <a href="http://www.ncbi.nlm.nih.gov/structure/?term=Karle,%20I">NCBI's Molecular Modeling Database</a> and the <a href="http://www.rcsb.org/pdb/results/results.do?qrid=BA20E662&amp;tabtoshow=Current">PDB</a> structure databases only gave me a few structures –all from her husband. Searching with her maiden name was futile as well.</p> <p>I went back to scanning her papers.</p> <p>Titles like "Crystal structure of ..." were so tantalizing. Being involved in genomics for so long, I couldn't imagine how a journal like PNAS could publish a 2004 paper, with a title like "<em>Crystal and molecular structure of a benzo[a]pyrene 7,8-diol 9,10-epoxide N2-deoxyguanosine adduct: absolute configuration and conformation</em>"  without requiring the authors to deposit the structure data in a public database.</p> <p>Luckily, they did.  But the data weren't in the PDB or the NCBI.  Karle deposited her data in a database I'd never heard of, <a href="http://www.ccdc.cam.ac.uk">The Cambridge Crystallographic Data Centre (CCDC)</a>.  It still wasn't easy to find her structures, but I could do so if I  looked for an ID in the paper and used it to search.   In the case of valinomycin (above), I only knew about it from Wikipedia, and was able to search for it by name and get it from ChemSpider.</p> <p>Some additional steps were required to convert structures into a PDB format for viewing, but the structures could be displayed.</p> <p>It's nice to know that someone so productive left some kind of data behind.</p> <p>References:</p> <ol><li>Wikipedia, <a href="https://en.wikipedia.org/wiki/Isabella_Karle">Isabella Karle</a>,  accessed Dec. 1, 2015.</li> <li>Valinomycin, downloaded from <a href="http://www.chemspider.com/Chemical-Structure.21493802.html?rid=857f68e8-4b3d-4b08-beb1-132724bf3d13">ChemSpider</a>, Dec. 1, 2015</li> <li>Valinomycin, <a href="https://pubchem.ncbi.nlm.nih.gov/compound/5649#section=Top">PubChem record</a>.  Dec. 1, 2015.</li> </ol></div> <span><a title="View user profile." href="/author/sporte">sporte</a></span> <span>Tue, 12/01/2015 - 15:28</span> Tue, 01 Dec 2015 20:28:27 +0000 sporte 69977 at https://scienceblogs.com Something scary for Halloween - polio virus https://scienceblogs.com/digitalbio/2015/10/28/something-scary-for-halloween-polio-virus <span>Something scary for Halloween - polio virus</span> <div class="field field--name-body field--type-text-with-summary field--label-hidden field--item"><p>When my parents were young, summer made cities a scary place for young families.  My mother tells me children were often sent away from their homes to relatives in the country, if possible, and swimming pools were definitely off limits.  The disease they feared, poliomyelitis, and the havoc it wrecked were the stuff of nightmares.  Children could wake up with a headache and end up a few hours later, in an iron lung, struggling to breathe.</p> <div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/10/molecule_polio_MW.png"><img class="size-large wp-image-1168" src="http://scienceblogs.com/digitalbio/files/2015/10/molecule_polio_MW-400x385.png" alt="Poliovirus colored by molecule in Molecule World." width="400" height="385" /></a> Poliovirus colored by molecule in Molecule World. </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>Today, on Jonas Salk's birthday, I read in the NPR blog Goats and Soda, that we're almost free of this scourge.  The two scientists who developed vaccines against polio, Jonas Salk and Alfred Sabin, gave mankind the tools, and several others put them to good use.  Be sure to check out <a href="http://www.npr.org/sections/goatsandsoda/2015/10/26/451908297/next-year-could-mark-the-end-of-polio" target="_blank">Jason Beaubien's article </a>and the amazing graphics showing poliovirus cases dropping across the world.</p> <p>Since we have the luxury of distance, vaccines, and molecular modeling apps, we can explore the scary poliovirus from the safety of a phone or iPad.  The way that I like to explore molecule models is to view a structure with different coloring styles and drawing styles and see if I can find patterns.</p> <p>To explore poliovirus, I opened Molecule World** and downloaded structure <a href="http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=34198" target="_blank">1XYR</a> (Bubeck, et. al) from the Molecular Modeling Database (MMDB) at the NCBI. The 1XYR model is from a form the virus takes when it's about to enter a cell.  It was obtained through cryo-electron microscopy.</p> <p>In the first image, each protein in the poliovirus capsid is shown in a different color.  The resolution isn't great, because electron microscopy can only do so much, so the structure model is built from the alpha carbon backbones of these 420 proteins.</p> <p>In the image below, I changed the drawing style to tubes and applied secondary structure coloring to make the next image.  Beta sheets are shown in orange and they make some interesting patterns on the surface of the capsid.</p> <div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/10/2nd_polio.png"><img class="size-large wp-image-1169" src="http://scienceblogs.com/digitalbio/files/2015/10/2nd_polio-400x388.png" alt="Poliovirus colored by secondary structure in Molecule World" width="400" height="388" /></a> Poliovirus colored by secondary structure in Molecule World </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>I also found, when using a neutral color and spinning the capsized around that there appeared to be some kind of ring or pore in the structure.  In the image below, I used a spacefill drawing style, colored by charge, and put the ring in the center.  The amino acids around the "opening" are grey, showing they're uncharged.  The images below that show beta sheets in the region around the ring and in residue coloring, the residue color key shows there are leucines, asparagine, and glycine.</p> <div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/10/poliovirus_charge_MW.png"><img class="size-large wp-image-1171" src="http://scienceblogs.com/digitalbio/files/2015/10/poliovirus_charge_MW-400x283.png" alt="Poliovirus colored by charge" width="400" height="283" /></a> Poliovirus colored by charge </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <div style="width: 410px;float:left;"><a href="/files/digitalbio/files/2015/10/poliovirus_2space_Mw.png"><img class="size-large wp-image-1172" src="http://scienceblogs.com/digitalbio/files/2015/10/poliovirus_2space_Mw-400x283.png" alt="Poliovirus drawn as spacefill, colored by secondary structure" width="400" height="283" /></a> Poliovirus drawn as spacefill, colored by secondary structure </div> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p><a href="/files/digitalbio/files/2015/10/polivirus_residue_MW.png"><img class="alignleft size-large wp-image-1173" src="http://scienceblogs.com/digitalbio/files/2015/10/polivirus_residue_MW-400x283.png" alt="polivirus_residue_MW" width="400" height="283" /></a></p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p> </p> <p>I'm not sure what this pore or ring structure means for the virus, but for me, it means I'll have to read the paper and find out.</p> <p>** I used the <a href="https://itunes.apple.com/us/app/molecule-world/id863565223?mt=8" target="_blank">iPad version of Molecule World</a>.  I think <a href="http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3dmac.shtml" target="_blank">Cn3D</a> might work, too, but I had some problems.  1XYR can also be viewed with the Molecule World for iPhone, but the screen will be black when the structure is downloaded.  Change the viewing mode to spacefill and you'll see the structure appear.</p> <p> </p> <p><strong>Reference</strong>:</p> <p>Bubeck D, Filman DJ, Cheng N, Steven AC, Hogle JM, Belnap DM. <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1143686/" target="_blank">The Structure of the Poliovirus 135S Cell Entry Intermediate at 10-Angstrom Resolution Reveals the Location of an Externalized Polypeptide That Binds to Membranes</a> . <i>Journal of Virology</i>. 2005;79(12):7745-7755. doi:10.1128/JVI.79.12.7745-7755.2005.</p> <p>Madej T, Lanczycki CJ, Zhang D, et al. <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965051/" target="_blank">MMDB and VAST+: tracking structural similarities between macromolecular complexes</a>. <i>Nucleic Acids Research</i>. 2014;42(Database issue):D297-D303. doi:10.1093/nar/gkt1208.</p> </div> <span><a title="View user profile." href="/author/sporte">sporte</a></span> <span>Wed, 10/28/2015 - 12:15</span> Wed, 28 Oct 2015 16:15:39 +0000 sporte 69976 at https://scienceblogs.com Making the most of Molecule of the Month with Molecule World https://scienceblogs.com/digitalbio/2015/09/08/making-the-most-of-molecule-of-the-month-with-molecule-world <span>Making the most of Molecule of the Month with Molecule World</span> <div class="field field--name-body field--type-text-with-summary field--label-hidden field--item"><p>We've been fans of the Molecule of the Month series by David Goodsell, for many years. Not only is Dr. Goodsell a talented artist but he writes very clear descriptions of the ways molecules like proteins, RNA, and DNA work together and function inside a cell.</p> <p>To learn about proteins and their activities, I like to go directly to the <a href="http://www.rcsb.org/pdb/101/motm_archive.do">Molecule of the Month</a> page, where I can find a list of articles organized by molecule type and name.  Many of these articles can also be downloaded in a PDF format.</p> <p>A really nice of his articles is that he includes PDB IDs for all the structures he discusses.  The PDB IDs make it easy to download and interact with the structures in Molecule World.</p> <p>Structures from the articles can be viewed on-line in Jmol, but we prefer a more interactive method where we can download the structures, view the sequences, and use the sequences to show / hide or modify parts of the structure in Molecule World.</p> <p>For example, say we want to know more about the genetic engineering tools Cascade and CRISPR.  These are pretty exciting because they allow scientists to engineer genomes with greater precision than ever before.  To learn more, we select the link to <a href="http://www.rcsb.org/pdb/101/motm.do?momID=181">Cascade and CRISPR</a> from the article list.</p> <p>The articles is great for understanding some of the back story.  To find the structures, we can search the web page with the term "PDB".  Every time the words "PDB entries" or "PDB entry" appear, they'll be followed by a PDB ID.  We can use those PDB IDs to get the structures from the NCBI or PDB databases.</p> <p>At the PDB site, those IDs <em>are</em> linked to database records where we can download the pdb files, but it's not always so easy to figure out which of the ten files is the one you should download.</p> <p>A simpler method, is to type the ID in Molecule World and touch the search button.  When the file name appears, touch the name of the file and the molecule file will be downloaded and opened.</p> <p><a href="/files/digitalbio/files/2015/09/adding-new-structures.png"><img class=" size-large wp-image-1150 aligncenter" src="http://scienceblogs.com/digitalbio/files/2015/09/adding-new-structures-400x368.png" alt="adding new structures" width="400" height="368" /></a></p> <p> </p> <p> </p> <p>For Cascade and CRISPR, the PDB IDs and structures in the article are listed below.  If you're using Molecule World on the iPhone or iPad, I set the links up so that touching the links will download the file and make it easy to open these in the app.</p> <p>All the images below were made in Molecule World.</p> <p>Two views of the cascade surveillance complex, a large complex of proteins and RNA  <a href="http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdb_strview.cgi/mmdb.cn3?program=cn3d&amp;display=0&amp;complexity=3&amp;buidx=1&amp;uid=122417">4TVX</a>, <a href="http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdb_strview.cgi/mmdb.cn3?program=cn3d&amp;display=0&amp;complexity=3&amp;buidx=1&amp;uid=122778">4U7U</a>,</p> <p><a href="/files/digitalbio/files/2015/09/4TVX.png"><img class=" size-large wp-image-1151 aligncenter" src="http://scienceblogs.com/digitalbio/files/2015/09/4TVX-400x509.png" alt="4TVX" width="400" height="509" /></a></p> <p><a href="/files/digitalbio/files/2015/09/4U7U.png"><img class=" size-large wp-image-1152 aligncenter" src="http://scienceblogs.com/digitalbio/files/2015/09/4U7U-400x488.png" alt="4U7U" width="400" height="488" /></a></p> <p>Cas1 and Cas2, proteins that chop up and store viral DNA <a href="http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdb_strview.cgi/mmdb.cn3?program=cn3d&amp;display=0&amp;complexity=3&amp;buidx=1&amp;uid=119621">4P6I</a>,</p> <p><a href="/files/digitalbio/files/2015/09/4P6I.png"><img class=" size-large wp-image-1153 aligncenter" src="http://scienceblogs.com/digitalbio/files/2015/09/4P6I-400x242.png" alt="4P6I" width="400" height="242" /></a></p> <p>Cas3, a nuclease that attacks viral DNA:  <a href="http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdb_strview.cgi/mmdb.cn3?program=cn3d&amp;display=0&amp;complexity=3&amp;buidx=1&amp;uid=122750">4QQW</a>,</p> <p><a href="/files/digitalbio/files/2015/09/4QQW1.png"><img class=" size-large wp-image-1155 aligncenter" src="http://scienceblogs.com/digitalbio/files/2015/09/4QQW1-400x534.png" alt="4QQW" width="400" height="534" /></a></p> <p> </p> <p>Cas9, a CRISPR system from another type of bacteria (<em>Streptococcus pyogenes</em>), with CRISPR RNA bound to target DNA <a href="http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdb_strview.cgi/mmdb.cn3?program=cn3d&amp;display=0&amp;complexity=3&amp;buidx=1&amp;uid=121809">4UN3</a></p> <p><a href="/files/digitalbio/files/2015/09/4UN3.png"><img class=" size-large wp-image-1156 aligncenter" src="http://scienceblogs.com/digitalbio/files/2015/09/4UN3-400x443.png" alt="4UN3" width="400" height="443" /></a></p> <p>And more complexes with both the CRISPR RNA and viral target DNA:  <a href="http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdb_strview.cgi/mmdb.cn3?program=cn3d&amp;display=0&amp;complexity=3&amp;buidx=1&amp;uid=117887">4OO8</a>,</p> <p><a href="/files/digitalbio/files/2015/09/4OO8.png"><img class=" size-large wp-image-1157 aligncenter" src="http://scienceblogs.com/digitalbio/files/2015/09/4OO8-400x416.png" alt="4OO8" width="400" height="416" /></a></p> <p>And our favorite,<a href="http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdb_strview.cgi/mmdb.cn3?program=cn3d&amp;display=0&amp;complexity=3&amp;buidx=1&amp;uid=122928">4QYZ</a>.</p> <p><a href="/files/digitalbio/files/2015/09/4QYZ.png"><img class=" size-large wp-image-1158 aligncenter" src="http://scienceblogs.com/digitalbio/files/2015/09/4QYZ-400x413.png" alt="4QYZ" width="400" height="413" /></a></p> <p>My favorite way to view some of these is to open the sequence viewer, find and select the nucleic sequences, and color by residue to distinguish between the RNA and DNA.  We made the RNA residues a little brighter since they're more chemically active.  It also helps to draw the models in different ways to see how the RNA is positioned inside the protein complex.</p> <p>To learn more about CRISPR and the Cascade proteins, iBiology has a <a href="https://vimeo.com/124545344">great video interview with Jennifer Doudna</a>, the woman who discovered this system.</p> </div> <span><a title="View user profile." href="/author/sporte">sporte</a></span> <span>Tue, 09/08/2015 - 16:52</span> Tue, 08 Sep 2015 20:52:00 +0000 sporte 69975 at https://scienceblogs.com