Biomedical subjects
Publications and source records attributed to Joseph A Mindell.
ClC chloride channels orchestrate the movement of chloride necessary for proper neuronal, muscular, cardiovascular, and epithelial function. In this issue of Neuron, Jentsch, Pusch, and colleagues use the structure of a bacterial ClC homolog to guide a mutagenic analysis of inhibitor binding to ClC-0, ClC-1, and ClC-2.
Accurate knowledge of defocus and tilt parameters is essential for the determination of three-dimensional protein structures at high resolution using electron microscopy. We present two computer programs, CTFFIND3 and CTFTILT, which determine defocus parameters from images of untilted specimens, as well as defocus and tilt parameters from images of tilted specimens, respectively. Both programs use a simple algorithm that fits the amplitude modulations visible in a power spectrum with a calculated contrast transfer function (CTF). The background present in the power spectrum is calculated using a low-pass filter. The background is then subtracted from the original power spectrum, allowing the fitting of only the oscillatory component of the CTF. CTFTILT determines specimen tilt parameters by measuring the defocus at a series of locations on the image while constraining them to a single plane. We tested the algorithm on images of two-dimensional crystals by comparing the results with those obtained using crystallographic methods. The images also contained contrast from carbon support film that added to the visibility of the CTF oscillations. The tests suggest that the fitting procedure is able to determine the image defocus with an error of about 10nm, whereas tilt axis and tilt angle are determined with an error of about 2 degrees and 1 degrees, respectively. Further tests were performed on images of single protein particles embedded in ice that were recorded from untilted or slightly tilted specimens. The visibility of the CTF oscillations from these images was reduced due to the lack of a carbon support film. Nevertheless, the test results suggest that the fitting procedure is able to determine image defocus and tilt angle with errors of about 100 nm and 6 degrees, respectively.
ClC-type Cl- channels, as two-pore homodimers, display an architecture unprecedented in selective ion channels, yet little is known regarding their mechanisms of selectivity and gating. In contrast to the great successes with K+ channels, a decade of mutagenic analysis has revealed little about the structure and function of the ClCs: even the number of ion-conducting pores per complex is controversial. Thus, for these proteins direct structural information is particularly important. We have formed two-dimensional crystals of a bacterial CIC homologue, and are analysing their structure using cryo-electron microscopy of glucose-embedded specimens. Here we report the measurement of electron diffraction patterns from these crystals. Unfettered by the imaging limitations of the electron microscope, the diffraction patterns reveal ordering of the crystals to at least 3.8 A resolution, suggesting that they can be used to generate an atomic model of the protein. We present an improved projection structure of the channel at 6.5 A using amplitude data derived from four electron diffraction patterns, with crystallographic statistics comparable to those reported for other high-quality two-dimensional crystals.