In x-ray crystallography, molecules are coaxed to form crystals, which are bombarded with intense beams of x-rays like those generated at the synchrotron beamlines seen here.
Image courtesy of Corie Ralston
Each atom in the crystal scatters the x-rays, producing a diffraction pattern like this one. Scientists gather diffraction data from many angles by rotating the crystal in the beam. With help from a high-powered computer, the data are combined with a known protein sequence to produce a three-dimensional map of the molecule.
Image courtesy of Jeff Dahl via Wikimedia Commons.
Electron microscopes are used in a variety of structural biology techniques.
Image courtesy of Pleple2000 via Wikimedia Commons.
The microscopes illuminate samples with electrons, producing high-magnification images of proteins like the human TFIID seen here. The technology requires only small amounts of sample and is well suited to visualizing larger molecular assemblies. Because the molecules aren’t locked into a crystal, it’s possible to capture the dynamics of very flexible proteins.
Image from Andel, F. et al. 1999. Science 286:2153-2156.
In cryoelectron microscopy, samples are flash frozen by plunging them into liquid nitrogen before going into the microscope. The freezing preserves a layer of water around the protein, capturing it in a more natural environment than that created by the harsh stains required in regular electron microscopy. HHMI Investigator Eva Nogales used cryoelectron microscopy to reveal the coexistence of these two distinct structures of TFIID, one of which is able to bind DNA.
Image courtesy of Michael Cianfrocco and Eva Nogales.
Electron crystallography is a mash-up of electron microscopy and x-ray crystallography. Two-dimensional protein crystals are exposed to an intense beam of electrons from an electron microscope. The resulting diffraction patterns are similar to those produced in x-ray crystallography. The technique is ideal for membrane proteins that cannot easily form large threedimensional crystals. JFRC Lab Head Tamir Gonen used electron crystallography to get this clear image of the two lipid bilayers (yellow) that surround the membrane protein aquaporin.
Image from Tamir Gonen and the Thomas Walz lab.
Nuclear magnetic resonance spectrometers expose molecules to a giant magnet that causes their atomic nuclei to absorb and re-emit electromagnetic radiation. The emitted energy gives clues about each atom’s orientation and location in the protein.
Image courtesy of Lihan Yao via Wikimedia Commons.
Because the molecules are free to move about, the technique is useful for watching molecules move and interact. NMR helped HHMI Investigator Dorothee Kern figure out which parts of the protein cyclophilin A were mobile (highlighted in red and blue).
Image from Eisenmesser, E.Z. et al. 2005. Nature 438:117–121.
In mass spectrometry, a molecule is sliced into pieces that are then ionized—electrons are added or removed to create charged particles. The fragments are sorted by their mass and charge, and then the fragmentation pattern is compared with patterns predicted for the protein to get an idea of which parts of a molecule are near each other.
Image courtesy of the Centre for High Throughput Biology (CHiBi), The University of British Columbia.
The technique also can be used to learn how molecules, like the ones in the type III secretion system, interact with each other by cross-linking the proteins—permanently binding the molecules together—and studying the resulting fragmentation pattern.
Image courtesy of the Strynadka Lab.
Instead of collaborating with other lab groups, some HHMI scientists have gathered the structural techniques they need right under their own roof. An x-ray crystallographer by training, Strynadka has added EM, cryo-EM, NMR, and customized mass spectrometry approaches to her own lab. She needs them all to study the membrane protein assemblies she is targeting for antibacterial and vaccine development.
