Hexbyte Glen Cove Solving a decades-old structural mystery surrounding the birth of energy-storing lipid droplets

Hexbyte Glen Cove

Depiction of the seipin complex from a top (above) and side view. The 10-unit seipin cage, made of A-shaped and B-shaped units, may become 10 A-shaped units right before and during budding of the lipid droplet. Triglycerides (yellow) are the fat component of the lipid droplet. Credit: UT Southwestern Medical Center

In humans, virtually every cell stores fat. However, patients with a rare condition called congenital lipodystrophy, which is often diagnosed in childhood, cannot properly store fat, which accumulates in the body’s organs and increases the risk of early death from heart or liver disease. In 2001, a transmembrane protein called seipin was identified as a molecule essential for proper fat storage, although its mechanism has remained unknown.

An international study published in Nature Structural & Molecular Biology is the first to solve and model virtually the entire structure of seipin, revealing it exists in two conformations and pointing to the mechanism for birthing the used for fat storage in healthy cells.

“Lipid droplets (LDs) have been described since the invention of microscopes that could show the inside of cells. For about a century, they’ve been known to store lipids, or fats, but they were considered inactive. During the past 20 years, lipid droplets have been shown to be very dynamic,” said Joel M. Goodman, Ph.D., Professor of Pharmacology at UT Southwestern, a Distinguished Teaching Professor, and one of the study’s three corresponding authors.

Dr. Goodman has played a key role in seipin biology, discovering in 2007 that seipin is responsible for packaging fat into LDs and that the same mechanism occurs in animals, plants, and fungi. In 2010, the Goodman lab was the first to purify seipin and reported that it was composed of about nine identical subunits that resembled a donut.

Ever since, scientists around the world had tried to solve the structure, which proved very difficult because seipin stretches across the membrane of the endoplasmic reticulum, an organelle within the cell. That transmembrane placement made the complex resistant to X-ray crystallography, the longtime gold standard for such studies. Membrane proteins are notoriously difficult to crystallize, a requirement for that technique.

To tackle the problem, Dr. Goodman turned to (cryo-EM) after discussions with Boston cell biologist Tobias C. Walther, Ph.D., at a scientific conference. Dr. Walther, a Howard Hughes Medical Institute Investigator, and his colleague, Robert V. Farese Jr., M.D., are the study’s other corresponding authors. They both have appointments at Harvard Medical School, the T.H. Chan School of Public Health, and the Broad Institute of MIT and Harvard. The study used the Harvard cryo-EM facility.

Cryo-EM uses flash-frozen samples, electron beams, and an electron detector rather than a camera to gather data on biological structures at near-atomic scale. Using cryo-EM enabled the researchers to determine that the “donut” they hypothesized was actually a 10-unit cage, a sort of incubator to create and grow lipid droplets. The second conformation showed seipin opening to release the lipid droplet onto the surface of the endoplasmic reticulum. Once on the surface, the LDs face the cell’s soupy interior (the cytoplasm), where passing enzymes can break down the LDs and free the fatty acids inside to provide energy such as during times of starvation, Dr. Goodman said.

“Getting two conformations was amazing, totally unexpected,” Dr. Goodman said, adding that previously other research teams had gotten a partial solution showing the lower layer of the seipin complex contained within the tube-like endoplasmic reticulum. The two conformations in the current investigation solve the elusive upper part of the structure, which extends across the organelle’s membrane.

“Cryo-EM made it possible,” Dr. Goodman said. “We hope that this structure will lead to a way of connecting seipin’s role in lipid-droplet creation to whatever goes wrong in lipodystrophy as well as help us better understand lipid-droplet formation in general,” he added. “There are likely several other proteins involved in the creation of lipid droplets, but seipin appears to be the main one. It seems to be a machine that generates lipid droplets.”

More information:
Henning Arlt et al, Seipin forms a flexible cage at lipid droplet formation sites, Nature Structural & Molecular Biology (2022). DOI: 10.1038/s41594-021-00718-y

Solving a decades-old structural mystery surrounding the birth of energy-storing lipid droplets (2022, February 25)
retrieved 28 February 2022
from https://phys.org/news/2022-02-decades-old-mystery-birth-energy-storing-lipid.html

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Hexbyte Glen Cove Solving a crystal’s structure when you’ve only got powder

Hexbyte Glen Cove

Artist’s rendition of X-ray beam illuminating a solution of powdered crystalline chalcogenates. Credit: Ella Maru Studios

Crystals reveal the hidden geometry of molecules to the naked eye. Scientists use crystals to figure out the atomic structure of new materials, but many can’t be grown large enough. Now, a team of researchers report a new technique in the January 19 issue of Nature that can discover the crystalline structure of any material.

To truly understand a chemical, a scientist needs to know how its atoms are arranged. Sometimes that’s easy: for example, both diamond and gold are made of a single kind of atom (carbon or gold, respectively) arranged in a cubic grid. But often it’s harder to figure out more complicated ones.

“Every single one of these is a special snowflake—growing them is really difficult,” says UConn chemical physicist Nate Hohman. Hohman studies metal organic chacogenolates. They’re made of a metal combined with an organic polymer and an element from column 16 of the periodic table (sulfur, selenium, tellurium or polonium.) Some are brightly colored pigments; others become more electrically conductive when light is shined on them; others make good solid lubricants that don’t burn up in the high temperatures of oil refineries or mines.

It’s a large, useful family of chemicals. But the ones Hohman studies—hybrid chalcogenolates—are really difficult to crystallize. Hohman’s lab couldn’t solve the atomic structures, because they couldn’t grow large perfect crystals. Even the tiny powdered crystals they could get were imperfect and messy.

X-ray crystallography is the standard way to figure out the atomic arrangements of more complicated materials. A famous, early example was how Rosalind Franklin used it to figure out the structure of DNA. She isolated large, perfect pieces of DNA in crystalline form, and then illuminated them with X-rays. X-rays are so small they diffract through the spaces between atoms, the same way visible light diffracts through slots in metal. By doing the math on the diffraction pattern, you can figure out the spacing of the slots—or atoms—that made it.

Once you know the atomic structure of a material, a whole new world opens up. Materials scientists use that information to design specific materials to do special things. For example, maybe you have a material that bends light in cool ways, so that it becomes invisible under ultraviolet light. If you understand the atomic structure, you might be able to tweak it—substitute a similar element of a different size in a specific spot, say—and make it do the same thing in visible light. Voila, an invisibility cloak!

Hybrid chalcogenolates, the compounds Hohman studies, won’t make you invisible. But they might make excellent new chemical catalysts and semiconductors. Currently he’s working with ones based on silver. His favorite, mithrene, is made of silver and selenium and glows a brilliant blue in UV light or “whenever grad students are around,” Hohman says.

Elyse Schreiber, a chemistry graduate student in Hohman’s lab, convinced Hohman they should try illuminating some of the small, messy hybrid chalcogenolates in a high powered X-ray beam anyway. If they could figure out the math, it would solve all their problems.

While working at the Linac Coherent Light Source at the SLAC linear accelerator in Menlo Park, California, Schreiber met Aaron Brewster, a researcher at Berkeley. Brewster mentioned he’d solved the math required to solve the crystal structure of difficult materials using X-ray crystallography. But he needed something to test it on. Hohman and Schreiber had the material. They provided plenty of tiny, imperfect chalcogenolate crystals, which they mixed into water emulsified with Dawn dish soap (another indispensable item in Hohman’s lab that glows blue) and shot jets of them into the accelerator beam. Each X-ray pulse illuminated the crystals incredibly brightly, allowing Brewster to capture a snapshot of the atomic structures of hundreds of tiny crystals. With enough snapshots, Brewster was able to run the calculations and figure out how the atoms were arranged.

Not only did they solve the crystal structures—they also figured out that the previous best guesses of what those structures were had been wrong. In theory, the technique, called small-molecule serial femtosecond crystallography, or smSFX, can be used for any chemical or material.

Computer scientists Nicolas Sauter and Daniel Paley at Lawrence Berkeley National Laboratory also helped develop smSFX. When you have a true powder, Paley explains, it’s like having a million crystals that are all jumbled together, full of imperfections, and scrambled in every possible orientation. Rather than diffracting the whole jumble together and getting a muddied readout of electron densities, like existing powder diffraction techniques, smSFX is so precise that it can diffract individual grains, one at a time. “This gives it a special sharpening effect,” he said. “So that is actually the kind of secret sauce of this whole method. Normally you shoot all million at once, but now you shoot 10,000 all in sequence,” Paley says.

“There is a huge array of fascinating physical and even chemical dynamics that occur at ultrafast timescales and this technique could help us to understand how these dynamic events affect the structure of microcrystalline materials. In a way, connecting the dots between a material’s structure and its function,” Schreiber elaborates. Hohman is equally excited about their success.

“Now that we can solve these hard to crystallize structures, we can design the best” structures for our purposes, Hohman says. Often, a material will come close to having a certain desirable property, but its crystalline won’t be quite right. Hohman hopes that with the data they can get from X-ray crystallography using Brewster’s technique, they can design better materials from the ground up.

Now, Hohman and Brewster are collaborating with Tess Smidt, a machine learning specialist at MIT, to try to teach a computer to design materials with specific properties.

This work involved the use of the SACLA free-electron laser in Japan, the Linac Coherent Light Source at SLAC National Accelerator Laboratory, and the Molecular Foundry and National Energy Research Scientific Computing Centers, U.S. Department of Energy Office of Science user facilities located at Berkeley Lab.

More information:
Elyse Schriber, Chemical crystallography by serial femtosecond X-ra

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Hexbyte Glen Cove Solving a long-standing mystery about the desert's rock art canvas thumbnail

Hexbyte Glen Cove Solving a long-standing mystery about the desert’s rock art canvas

Hexbyte Glen Cove

Petroglyphs at Mesa Verde National Park, Colorado. Credit: Christine Fry & Peter Russo

Wander around a desert most anywhere in the world, and eventually you’ll notice dark-stained rocks, especially where the sun shines most brightly and water trickles down or dew gathers. In some spots, if you’re lucky, you might stumble upon ancient art—petroglyphs—carved into the stain. For years, however, researchers have understood more about the petroglyphs than the mysterious dark stain, called rock varnish, in which they were drawn.

In particular, science has yet to come to a conclusion about where , which is unusually rich in manganese, comes from.

Now, scientists at the California Institute of Technology, the Department of Energy’s SLAC National Accelerator Laboratory and elsewhere think they have an answer. According to a recent paper in Proceedings of the National Academy of Sciences, rock varnish is left behind by microbial communities that use manganese to defend against the punishing desert sun.

The mystery of rock varnish is old, said Usha Lingappa, a graduate student at Caltech and the study’s lead author. “Charles Darwin wrote about it, Alexander von Humboldt wrote about it,” she said, and there is a long-standing debate about whether it has a biological or inorganic origin.

But, Lingappa said, she and her colleagues didn’t actually set out to understand where rock varnish comes from. Instead, they were interested in how microbial ecosystems in the desert interact with rock varnish. To do so, they deployed as many techniques as they could come up with: DNA sequencing, mineralogical analyses, , and—aided by Stanford Synchroton Radiation Lightsource (SSRL) scientist Samuel Webb—advanced X-ray spectroscopy methods that could map different kinds of manganese and other elements within samples of rock varnish.

“By combining these different perspectives, maybe we could draw a picture of this ecosystem and understand it in new ways,” Lingappa said. “That’s where we started, and then we just stumbled into this hypothesis” for rock varnish formation.

Among the team’s key observations was that, while manganese in desert dust is usually in particle form, it was deposited in more continuous layers in varnish, a fact revealed by X-ray spectroscopy methods at SSRL that can tell not only what make up a sample but also how they are distributed, on a microscopic scale, throughout the sample.

That same analysis showed that the kinds of manganese compounds in varnish were the result of ongoing chemical cycles, rather than being left out in the sun for millennia. That information, combined with the prevalence of bacteria called Chroococcidiopsis that use manganese to combat the oxidative effects of the harsh desert sun, led Lingappa and her team to conclude that rock varnish was left behind by those bacteria.

For his part, Webb said that he always enjoys a project—”I’ve been a mangaphile for a while now”—and that this project arrived at the perfect time, given advances in X-ray spectroscopy at SSRL. Improvements in X-ray beam size allowed the researchers to get a finer-grained picture of rock varnish, he said, and other improvements ensured that they could get a good look at their samples without the risk of damaging them. “We’re always tinkering and fine-tuning things, and I think it was the right time for a project that maybe 5 or 10 years ago wouldn’t really have been feasible.”

More information:
Usha F. Lingappa et al, An ecophysiological explanation for manganese enrichment in rock varnish, Proceedings of the National Academy of Sciences (2021). DOI: 10.1073/pnas.2025188118

Solving a long-standing mystery about the desert’s rock art canvas (2021, July 2)
retrieved 3 July 2021
from https://phys.org/news/2021-07-long-standing-mystery-art-canvas.html

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