Imagem 3-D de uma proteína mostrando sua estrutura

sexta-feira, janeiro 27, 2012

Under the Electron Microscope: 3-D Image of an Individual Protein Showing Structure

ScienceDaily (Jan. 25, 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.


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) (Credit: Image courtesy of DOE/Lawrence Berkeley National Laboratory) 

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.

Scientists routinely create models of proteins using X-ray diffraction, nuclear magnetic resonance, and conventional cryo-electron microscope (cryoEM) imaging. But these models require computer "averaging" of data from analysis of thousands, or even millions of like molecules, because it is so difficult to resolve the features of a single particle. Ren and Zhang have done just that, generating detailed models using electron microscopic images of a single protein.

He calls his technique "individual-particle electron tomography," or IPET. The work is described in the January 24 issue of PLoS ONE, the open-source scientific journal.

The 3-D images reported in the paper include those of a single IgG antibody and apolipoprotein A-1 (ApoA-1), a protein involved in human metabolism. Ren's goal is to produce individual 3-D images of medically significant proteins, such as HDL -- the heart-protective "good cholesterol" whose structure has eluded the efforts of legions of scientists armed with far more powerful protein modeling tools. "We are well on our way," says Ren.

Ren has the credentials of one who knows what he can do. He was recruited to work at Berkeley Lab in August 2010 from the University of California at San Francisco, where he had used a cryo-electron microscope and more conventional averaging techniques to discern the 3-D structure of LDL -- the "bad cholesterol" thought to be a major risk factor for heart disease.

His images of single proteins are a bit fuzzy, even after they are cleaned up by complex computer filtering, but very informative to the trained observer. These individual particles are extraordinarily tiny, requiring Ren to zero in on a spot of less than 20 nanometers. He has reported protein images as small as 70 kDa. That's kilodaltons, a Lilliputian scale (expressed in units of mass) set aside for taking the measure of atoms, molecules, and snippets of DNA. It's a more useful way to size soft objects like proteins that can be clumped, stringy, or floppy.
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Read more here/Leia mais aqui: Science Daily

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IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure

Lei Zhang, Gang Ren*

Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America

Abstract 

The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single “snapshot” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.

Citation: Zhang L, Ren G (2012) IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure. PLoS ONE 7(1): e30249. doi:10.1371/journal.pone.0030249

Editor: Wenqing Xu, University of Washington, United States of America

Received: July 6, 2011; Accepted: December 14, 2011; Published: January 24, 2012

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Funding: This work was supported by the Office of Science, Office of Basic Energy Sciences of the United States Department of Energy (contract no. DE-AC02-05CH11231) and partially supported by the William Myron Keck Foundation (#011808). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: gren@lbl.gov

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