Large protein assemblies such as the nuclear pore complex (NPC), a channel that consists of several hundred proteins, are critical to cell function. But due their size, these structures have typically been difficult to analyze. However, Jan Ellenberg and colleagues at EMBL recently published a paper in Science describing a new technique they used to determine the structure of the NPC—a technique that can be applied to other similarly-sized, complex molecular assemblies, such as the kinetochore, the centrosome, and coated vesicles.
Ellenberg’s group started with stochastic super-resolution microscopy, a technique that can generate a resolution of 15 nanometers, depending on how precisely the fluorophores are located and how completely the structure is labeled. By combining the information from several thousand images taken with stochastic super-resolution microscopy, the scientists were able to precisely determine a partial structure of the complex. “The basic idea is that the more structures you take into your measurement, the better your statistics become, and the more precise you can be,” explained Anna Szymborska, one of the co-authors of the paper.
The team created an average image from which they could read very high-precision data. The first step was labeling the different proteins that form the NPC. With the super-resolution microscope alone, they were able to see that the NPC formed a ring-like structure. That gave them enough spatial information to align the images with each other.
“We very precisely centered the images of one type of label on the pore, then summed them up to generate the average image,” said Szymborska. From the fluorescence profile of the average image, they were able to figure out where the label generating that particular signal was located respective to the pore’s center.
Szymborska said the method can be applied to other large protein complexes in the cell. Although their alignment of the NPC relied on the fact that it was a symmetrical structure, the same method can be extended to asymmetric structures by aligning to a molecular reference labeled with a different color marker and measuring the distance to this reference signal.
Next, they’d like to apply the method to 3-D analysis of the NPC. “This study was very simplified because we asked a very targeted question about how something is oriented in the pore, but we would like to understand the whole structure more thoroughly,” said Szymborska.
1. Szymborska, A., A. de Marco, N. Daigle, V.C. Cordes, J.A. Briggs, J. Ellenberg. 2013. Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging. Science (July).