{"id":13998,"date":"2017-07-03T00:00:17","date_gmt":"2017-07-03T07:00:17","guid":{"rendered":"https:\/\/vermont.salk.edu\/?post_type=disclosure&p=13998"},"modified":"2024-01-30T15:32:11","modified_gmt":"2024-01-30T23:32:11","slug":"tilted-microscopy-technique-better-reveals-protein-structures","status":"publish","type":"disclosure","link":"https:\/\/www.salk.edu\/zh\/news-release\/tilted-microscopy-technique-better-reveals-protein-structures\/","title":{"rendered":"Tilted microscopy technique better reveals protein structures"},"content":{"rendered":"

LA JOLLA\u2014The conventional way of placing protein samples under an electron microscope during cryo-EM experiments may fall flat when it comes to getting the best picture of a protein\u2019s structure. In some cases, tilting a sheet of frozen proteins\u2014by anywhere from 10 to 50 degrees\u2014as it lies under the microscope, gives higher quality data and could lead to a better understanding of a variety of diseases, according to new research led by Salk scientist Dmitry Lyumkis<\/a>.<\/p>\n

\"\"
Just as looking at soup cans from different angles allows you to see different shapes, viewing proteins at a tilt reveals different aspects of their structure. <\/p>\n

Click here<\/a> for a high-resolution image. <\/p>\n

Credit: Salk Institute<\/figcaption><\/figure>\n

\u201cPeople have tried to implement tilting before, but there have been a lot of challenges,\u201d says Lyumkis, a Helmsley-Salk Fellow at the Salk Institute and senior author of the new work, published July 3, 2017, in Nature Methods<\/em><\/a>. \u201cWe\u2019ve eliminated many of these problems with our new approach.\u201d<\/p>\n

Cryo-EM, or cryo-electron microscopy, is a form of transmission electron microscopy in which samples are quickly cooled to below freezing before being imaged under the microscope. Unlike other methods commonly used to determine the structure of proteins, cryo-EM lets proteins remain in their natural conformations for imaging, which could reveal new information about the structures. Understanding proteins\u2019 structures is a vital step to developing new therapies for disease, such as in the case of HIV<\/a>.<\/p>\n

Researchers have long assumed that proteins adopt random conformations throughout the frozen grid that\u2019s prepared for cryo-EM experiments, which means that by taking enough images, researchers can put together a full, 3D picture of the protein\u2019s shape(s) from all imaging directions. But for many proteins, the approach seems to fall short, and parts of the proteins\u2019 structures remain missing.<\/p>\n

\u201cResearchers are starting to think that the proteins on a cryo-EM grid don\u2019t adopt random conformations after all, but rather stick to the top or bottom of the sample grid in preferred orientations,\u201d says Lyumkis. \u201cThus we may not be getting the full picture of proteins\u2019 structures. More importantly, this behavior can prohibit structure determination altogether for select protein samples.\u201d<\/p>\n

To understand the problem, imagine trying to look at the shadows of a dozen tin cans to figure out their shape but seeing only circles because all the cans are exactly upright. By making the light\u2014or electron beam, in the case of cryo-EM\u2014hit the samples at an angle, though, you\u2019d be able to see the true shape better.<\/p>\n

When researchers have tried to tilt samples under a microscope in the past, they\u2019ve been limited by poor resolution: an angle means that the electron beam has to travel through a thicker grid. Samples are also more likely to move within the frozen grid when they\u2019re tilted, blurring out the data. And technically, analyzing data from a tilted sample is also more challenging, since cryo-EM methods were designed with the assumption that the grid containing proteins was always at the same distance from the microscope.<\/p>\n

\"Dmitry
Dmitry Lyumkis<\/p>\n

Click here<\/a> for a high-resolution image.<\/p>\n

Credit: Salk Institute<\/figcaption><\/figure>\n

To tackle these challenges, Lyumkis and his colleagues changed the materials used to create the cryo-EM grid, recorded movies of their data rather than still images, and developed new computational methods to analyze the information.<\/p>\n

When they tested the new approach on the influenza hemagglutinin protein, a notoriously hard protein to characterize using cryo-EM, the team found that tilting the sample gave a more complete dataset. When the protein sample was flat, typical algorithms introduced false positive shape to the protein that wasn\u2019t backed up by experimental data. That wasn\u2019t the case when it was tilted.<\/p>\n

\u201cDue to the geometry of the data collection when we tilt, we fill up much more data characterizing the molecules, giving us a more complete picture of the protein\u2019s shape\u201d says Lyumkis.<\/p>\n

The algorithms that Lyumkis and his team developed\u2014which include ways to analyze whether a cryo-EM experiment is introducing bad data, as well as the methods to interpret a tilted experiment\u2014are now openly available. They hope other researchers will start using them and that it becomes a standard metric for cryo-EM structure validation (since most experimentally derived structures suffer from missing information to different extents).<\/p>\n

\u201cOne of the ideas we\u2019re looking at now is whether data collection should always be performed at a tilt rather than in the conventional way,\u201d says Lyumkis. \u201cIt won\u2019t hurt and it should help.\u201d<\/p>\n

Other researchers on the study were Yong Zi Tan, Philip Baldwin, Clinton Potter and Bridget Carragher of the New York Structural Biology Center<\/a>, and Joseph David and James Williamson of The Scripps Research Institute<\/a>.<\/p>\n

The work and the researchers involved were supported by grants from the Agency for Science, Technology, and Research Singapore<\/a>, the Leona M. and Harry B. Helmsley Charitable Trust<\/a>, the U.S. National Institutes of Health<\/a>, the Jane Coffin Childs Foundation<\/a>, the National Institute of Aging<\/a>, the National Institute of General Medical Sciences<\/a> and the Simons Foundation<\/a>.<\/p>","protected":false},"featured_media":14011,"template":"","faculty":[320],"disease-research":[122,331],"class_list":["post-13998","disclosure","type-disclosure","status-publish","has-post-thumbnail","hentry","faculty-dmitry-lyumkis","disease-research-immune-system-biology","disease-research-protein-interactions"],"acf":[],"yoast_head":"\nTilted microscopy technique better reveals protein structures - Salk Institute for Biological Studies<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.salk.edu\/zh\/news-release\/tilted-microscopy-technique-better-reveals-protein-structures\/\" \/>\n<meta property=\"og:locale\" content=\"zh_CN\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Tilted microscopy technique better reveals protein structures - Salk Institute for Biological Studies\" \/>\n<meta property=\"og:description\" content=\"LA JOLLA\u2014The conventional way of placing protein samples under an electron microscope during cryo-EM experiments may fall flat when it comes to getting the best picture of a protein\u2019s structure. 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