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  • richardmitnick 8:00 am on March 31, 2016 Permalink | Reply
    Tags: , , , LBL Molecular Foundry,   

    From LBL: “Revealing the Fluctuations of Flexible DNA in 3-D” 

    Berkeley Logo

    Berkeley Lab

    March 30, 2016
    Glenn Roberts Jr.
    510-486-5582
    geroberts@lbl.gov

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    In a Berkeley Lab-led study, flexible double-helix DNA segments connected to gold nanoparticles are revealed from the 3-D density maps (purple and yellow) reconstructed from individual samples using a Berkeley Lab-developed technique called individual-particle electron tomography or IPET. Projections of the structures are shown in the background grid. (Credit: Berkeley Lab)

    An international team working at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) has captured the first high-resolution 3-D images from individual double-helix DNA segments attached at either end to gold nanoparticles. The images detail the flexible structure of the DNA segments, which appear as nanoscale jump ropes.

    This unique imaging capability, pioneered by Berkeley Lab scientists, could aid in the use of DNA segments as building blocks for molecular devices that function as nanoscale drug-delivery systems, markers for biological research, and components for computer memory and electronic devices. It could also lead to images of important disease-relevant proteins that have proven elusive for other imaging techniques, and of the assembly process that forms DNA from separate, individual strands.

    The shapes of the coiled DNA strands, which were sandwiched between polygon-shaped gold nanoparticles, were reconstructed in 3-D using a cutting-edge electron microscope technique coupled with a protein-staining process and sophisticated software that provided structural details to the scale of about 2 nanometers, or two billionths of a meter.

    “We had no idea about what the double-strand DNA would look like between the nanogold particles,” said Gang “Gary” Ren, a Berkeley Lab scientist who led the research. “This is the first time for directly visualizing an individual double-strand DNA segment in 3-D,” he said. The results were published in the March 30 edition of Nature Communications.

    The method developed by this team, called individual-particle electron tomography (IPET), had earlier captured the 3-D structure of a single protein that plays a key role in human cholesterol metabolism. By grabbing 2-D images of the same object from different angles, the technique allows researchers to assemble a 3-D image of that object. The team has also used the technique to uncover the fluctuation of another well-known flexible protein, human immunoglobulin 1, which plays a role in our immune system.


    Access mp4 video here .

    For this latest study of DNA nanostructures, Ren used an electron-beam study technique called cryo-electron microscopy (cryo-EM) to examine frozen DNA-nanogold samples, and used IPET to reconstruct 3-D images from samples stained with heavy metal salts. The team also used molecular simulation tools to test the natural shape variations, called “conformations,” in the samples, and compared these simulated shapes with observations.

    Ren explained that the naturally flexible dynamics of samples, like a man waving his arms, cannot be fully detailed by any method that uses an average of many observations.

    A popular way to view the nanoscale structural details of delicate biological samples is to form them into crystals and zap them with X-rays, though this does not preserve their natural shape and the DNA-nanogold samples in this study are incredibly challenging to crystallize. Other common research techniques may require a collection of thousands near-identical objects, viewed with an electron microscope, to compile a single, averaged 3-D structure. But this 3-D image may not adequately show the natural shape fluctuations of a given object.

    The samples in the latest experiment were formed from individual polygon gold nanostructures, measuring about 5 nanometers across, connected to single DNA-segment strands with 84 base pairs. Base pairs are basic chemical building blocks that give DNA its structure. Each individual DNA segment and gold nanoparticle naturally zipped together with a partner to form the double-stranded DNA segment with a gold particle at either end.


    Access mp4 video here .
    These views compare the various shape fluctuations obtained from different samples of the same type of double-helix DNA segment (DNA renderings in green, 3-D reconstructions in purple) connected to gold nanoparticles (yellow). (Credit: Berkeley Lab)

    The samples were flash-frozen to preserve their structure for study with cryo-EM imaging, and the distance between the two gold particles in individual samples varied from 20-30 nanometers based on different shapes observed in the DNA segments. Researchers used a cryo-electron microscope at Berkeley Lab’s Molecular Foundry for this study.

    They collected a series of tilted images of the stained objects, and reconstructed 14 electron-density maps that detailed the structure of individual samples using the IPET technique. They gathered a dozen conformations for the samples and found the DNA shape variations were consistent with those measured in the flash-frozen cryo-EM samples. The shapes were also consistent with samples studied using other electron-based imaging and X-ray scattering methods, and with computer simulations.

    While the 3-D reconstructions show the basic nanoscale structure of the samples, Ren said that the next step will be to work to improve the resolution to the sub-nanometer scale.

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    Gang Ren (standing) and Lei Zhang participated in a study at Berkeley Lab’s Molecular Foundry that produced 3-D reproductions of individual samples of double-helix DNA segments attached to gold nanoparticles. (Photo by Roy Kaltschmidt/Berkeley Lab)

    “Even in this current state we begin to see 3-D structures at 1- to 2-nanometer resolution,” he said. “Through better instrumentation and improved computational algorithms, it would be promising to push the resolution to that visualizing a single DNA helix within an individual protein.”

    The technique, he said, has already excited interest among some prominent pharmaceutical companies and nanotechnology researchers, and his science team already has dozens of related research projects in the pipeline.

    In future studies, researchers could attempt to improve the imaging resolution for complex structures that incorporate more DNA segments as a sort of “DNA origami,” Ren said. Researchers hope to build and better characterize nanoscale molecular devices using DNA segments that can, for example, store and deliver drugs to targeted areas in the body.

    “DNA is easy to program, synthesize and replicate, so it can be used as a special material to quickly self-assemble into nanostructures and to guide the operation of molecular-scale devices,” he said. “Our current study is just a proof of concept for imaging these kinds of molecular devices’ structures.”

    The Molecular Foundry is a DOE Office of Science User Facility.

    In addition to Berkeley Lab scientists, other researchers contributing to this study were from UC Berkeley, the Kavli Energy NanoSciences Institute at Berkeley Lab and UC Berkeley, and Xi’an Jiaotong University in China.

    This work was supported by the National Science Foundation, DOE Office of Basic Energy Sciences, National Institutes of Health, the National Natural Science Foundation of China, Xi’an Jiaotong University in China, and the Ministry of Science and Technology in China.

    View more about Gary Ren’s research group here.

    See the full article here .

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  • richardmitnick 12:41 pm on September 6, 2012 Permalink | Reply
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    From Berkeley Lab: “Forcing the Molecular Bond Issue” 


    Berkeley Lab

    New and Improved Model of Molecular Bonding from Researchers at Berkeley Lab’s Molecular Foundry

    September 05, 2012
    Lynn Yarris

    Material properties and interactions are largely determined by the binding and unbinding of their constituent molecules, but the standard model used to interpret data on the formation and rupturing of molecular bonds suffers from inconsistencies. A collaboration of researchers led by a scientist at the U.S Department of Energy (DOE)’s Lawrence Berkeley National Laboratory (Berkeley Lab) has developed a first-of-its-kind model for providing a comprehensive description of the way in which molecular bonds form and rupture. This model enables researchers to predict the ‘binding free energy’ of a given molecular system, which is key to predicting how that molecule will interact with other molecules.

    image
    Under dynamic force spectroscopy, the bonds of a molecular system are subjected to controlled stretching until the bonds break. (Image courtesy of Jim DeYoreo, Berkeley Lab)

    ‘Molecular binding and unbinding events are much simpler than we have been led to believe from the standard model over the past decade,’ says Jim DeYoreo, a scientist with the Molecular Foundry, a DOE nanoscience center at Berkeley Lab who was one of the leaders of this research. ‘With our new model, we now have a clear means for measuring one of the most important parameters governing how materials and molecules bind together.'”

    See the full article here.

    A U.S. Department of Energy National Laboratory Operated by the University of California

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  • richardmitnick 7:46 pm on February 21, 2012 Permalink | Reply
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    From Berkeley Lab: “How Good Cholesterol Turns Bad” 


    Berkeley Lab

    Berkeley Lab Researchers Find New Evidence on How Cholesterol Gets Moved from HDLs to LDLs

    February 21, 2012
    Lynn Yarris

    “Researchers with the U.S. Department of Energy (DOE)’s Lawrence Berkeley National Laboratory (Berkeley Lab) have found new evidence to explain how cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol from ‘good’ high density lipoproteins (HDLs) to ‘bad’ low density lipoproteins (LDLs). These findings point the way to the design of safer, more effective next generation CETP inhibitors that could help prevent the development of heart disease.

    Gang Ren, a materials physicist and electron microscopy expert with Berkeley Lab’s Molecular Foundry, a DOE nanoscience research center, led a study in which the first structural images of CETP interacting with HDLs and LDLs were recorded. The images and structural analyses support the hypothesis that cholesterol is transferred from HDLs to LDLs via a tunnel running through the center of the CETP molecule.

    ‘Our images show that CETP is a small (53 kilodaltons) banana-shaped asymmetric molecule with a tapered N-terminal domain and a globular C-terminal domain,’ Ren says. ‘We discovered that the CETP’s N-terminal penetrates HDL and its C-terminal interacts with LDL forming a ternary complex. Structure analyses lead us to hypothesize that the interaction may generate molecular forces that twist the terminals, creating pores at both ends of the CETP. These pores connect with central cavities in the CETP to form a tunnel that serves as a conduit for the movement of cholesterol from the HDL.'”

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    (1) CETP penetrates HDL to its cholesterol core. (2) Upon interaction with LDL/VLDL, molecular forces cause the formation of pores at either end of CETP. (3) These pores connect with CETP’s central cavities to form a tunnel for the transfer of cholesterol to LDL/VLDL, which (4) reduces HDL in size. (No image credit)

    See the full and very important article here.

    A U.S. Department of Energy National Laboratory Operated by the University of California

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  • richardmitnick 5:58 am on January 25, 2012 Permalink | Reply
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    From Berkeley Lab: “Under the Electron Microscope – A 3-D Image of an Individual Protein” 


    Berkeley Lab

    The high resolution of Lawrence Berkeley National Laboratory’s Gang Ren

    Sabin Russell
    JANUARY 24, 2012

    “When Gang Ren whirls the controls of his cryo-electron microscope, he compares it to fine-tuning the gearshift and brakes of a racing bicycle. But this machine at the U.S. Department of Energy (DOE)’s Lawrence Berkeley National Laboratory (Berkeley Lab) is a bit more complex. It costs nearly $1.5 million, operates at the frigid temperature of liquid nitrogen, and it is allowing scientists to see what no one has seen before.

    At the Molecular Foundry, Berkeley Lab’s acclaimed nanotechnology research center, Ren has pushed his Zeiss Libra 120 Cryo-Tem microscope to resolutions never envisioned by its German manufacturers, producing detailed snapshots of individual molecules. Today, he and his colleague Lei Zhang are reporting the first 3-D images of an individual protein ever obtained with enough clarity to determine its structure.”

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    3-D images from a single particle (A) a series of images of an ApoA-1 protein particle, taken from different angles as indicated. A succession of four computer enhancements (projections) clarifies the signal. In the right column is the 3-D image compiled from the clarified data. B) is a close-up of the reconstructed 3-D image. C) Analysis shows how the particle structure is formed by three ApoA-1 proteins (red, green, blue noodle-like models)

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    Staff scientist Gang Ren (standing) and is postdoc colleague Lei Zhang can checking images of individual proteins from their cryo-electron microscope at Berkeley Lab’s Molecular Foundry.

    See the full article here.

    A US Department of Energy National Laboratory Operated by the University of California

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