A 3D image of an individual protein

January 26, 2012

3-D images from a single particle: (A) a series of images of an ApoA-1 protein particle, taken from different angles. 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: Berkeley Lab)

Lawrence Berkeley National Laboratory (Berkeley Lab) scientists have created detailed models of a single protein using electron microscopic images.

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.

Gang Ren calls his technique “individual-particle electron tomography,” or IPET.

The 3-D images 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.

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.

Within the complex structure of these proteins lies the secrets of their function, and perhaps keys to drugs that block the bad ones and promote the good ones. With some additional computer filtering, a high-contrast model of protein can be generated from the images and animated to show its moving parts in 3-D.

By observing the structure of single proteins, it is possible to understand their flexible, moving parts. “This opens a door for the study of protein dynamics,” Ren says. “Antibodies, for example, are not solid. They are very flexible, very dynamic.”

How did Ren coax so much versatility out of his Libra 120? He’s equipped the microscope with a $300,000 CCD camera, some powerful image-processing software, special contrasting agents, and a device called an “energy filter” that sifts through the digitized camera data and culls weak signals. The multiple angles used to create the 3-D portrait help resolve the faint molecular image.

Electron microscopes focus streams of electrons rather than light to see incredibly tiny things. The short wavelength of an electron beam enables much higher resolution and magnification than visible light. Powerful electron microscopes have been used for decades to probe materials at atomic-scale; and right next door to the Molecular Foundry is Berkeley Lab’s National Center for Electron Microscopy, which houses the most powerful microscopes in the world. The TEAM 0.5 microscope can distinguish objects as small as the radius of a hydrogen atom. But these heavyweight microscopes pull off this atomic-scale resolution with pulses of energy that would obliterate most soft biological proteins. The high power electron microscopes are used primarily for probing atomic structure of strong, solid materials, such as graphene.

Ren’s lab specializes in cryoEM, which examines objects frozen at -180 °C (-292 °F). A bath of liquid nitrogen flash-freezes samples so quickly that no ice crystals form. The extremely low temperature fixes the samples and prevents them from drying out in the vacuum needed for the electron scan. It creates conditions favorable for imaging at much lower doses of electrons — low enough to keep a single soft protein intact while more than 100 images are taken over a one-to-two hour period.

Ref.: Lei Zhang and Gang Ren, IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure, PLoS ONE, 2012; [DOI:10.1371/journal.pone.0030249]

A computer animation demonstrates the flexible dynamics — the moving parts — of human IgG antibody. 3-D images of two individual antibody particles (gray) were generated using EM tomography with IPET. The demonstration shows how the same molecular chains (red, orange, and green noodle-like models) of antibody particle #1 can fit precisely into particle #2, which was found under the microscope in an entirely different pose.