The potassium channel from Streptomyces lividans is an integral membrane protein with sequence similarity to all known K+ channels, particularly in the pore region. X-ray analysis with data to 3.2 angstroms reveals that four identical subunits create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow selectivity filter is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center of the bilayer. Main chain carbonyl oxygen atoms from the K+ channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K+ ions but not smaller Na+ ions. The selectivity filter contains two K+ ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive forces to overcome attractive forces between K+ ions and the selectivity filter. The architecture of the pore establishes the physical principles underlying selective K+ conduction.

The KirBac1.1 channel belongs to the inward-rectifier family of potassium channels. Here we report the structure of the entire prokaryotic Kir channel assembly, in the closed state, refined to a resolution of 3.65 angstroms. We identify the main activation gate and structural elements involved in gating. On the basis of structural evidence presented here, we suggest that gating involves coupling between the intracellular and membrane domains. This further suggests that initiation of gating by membrane or intracellular signals represents different entry points to a common mechanistic pathway.

Toxins from scorpion venom interact with potassium channels. Resin-attached, mutant K+ channels from Streptomyces lividans were used to screen venom from Leiurus quinquestriatus hebraeus, and the toxins that interacted with the channel were rapidly identified by mass spectrometry. One of the toxins, agitoxin2, was further studied by mutagenesis and radioligand binding. The results show that a prokaryotic K+ channel has the same pore structure as eukaryotic K+ channels. This structural conservation, through application of techniques presented here, offers a new approach for K+ channel pharmacology.

The development of serial crystallography has been driven by the sample requirements imposed by X-ray free-electron lasers. Serial techniques are now being exploited at synchrotrons. Using a fixed-target approach to highthroughput serial sampling, it is demonstrated that high-quality data can be collected from myoglobin crystals, allowing room-temperature, low-dose structure determination. The combination of fixed-target arrays and a fast, accurate translation system allows high-throughput serial data collection at high hit rates and with low sample consumption.

We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.

Potassium channels allow the selective flow of K(+) ions across membranes. In response to external gating signals, the potassium channel can move reversibly through a series of structural conformations from a closed to an open state. 2D crystals of the inwardly rectifying K(+) channel KirBac3.1 from Magnetospirillum magnetotacticum have been captured in two distinct conformations, providing "snap shots" of the gating process. Analysis by electron cryomicroscopy of these KirBac3.1 crystals has resulted in reconstructed images in projection at 9 A resolution. Kir channels are tetramers of four subunits arranged as dimers of dimers. Each subunit has two transmembrane helices (inner and outer). In one crystal form, the pore is blocked; in the other crystal form, the pore appears open. Modeling based on the KirBac1.1 (closed) crystal structure shows that opening of the ion conduction pathway could be achieved by bending of the inner helices and significant movements of the outer helices.

The advent of ultrafast highly brilliant coherent X-ray free-electron laser sources has driven the development of novel structure-determination approaches for proteins, and promises visualization of protein dynamics on sub-picosecond timescales with full atomic resolution. Significant efforts are being applied to the development of sample-delivery systems that allow these unique sources to be most efficiently exploited for high-throughput serial femtosecond crystallography. Here, the next iteration of a fixed-target crystallography chip designed for rapid and reliable delivery of up to 11 259 protein crystals with high spatial precision is presented. An experimental scheme for predetermining the positions of crystals in the chip by means of in situ spectroscopy using a fiducial system for rapid, precise alignment and registration of the crystal positions is presented. This delivers unprecedented performance in serial crystallography experiments at room temperature under atmospheric pressure, giving a raw hit rate approaching 100% with an effective indexing rate of approximately 50%, increasing the efficiency of beam usage and allowing the method to be applied to systems where the number of crystals is limited.

The transmembrane protein CD5, expressed on all T cells and the B1 subset of B cells, modulates antigen receptor-mediated activation. We used the yeast twohybrid system to identify proteins that interact with its cytoplasmic domain and play a role in CD5 proximal signaling events. We found that the ␤ subunit of the serine/threonine kinase casein kinase 2 (CK2) interacts specifically with the cytoplasmic domain of CD5. Coimmunoprecipitation experiments showed activationindependent association of CK2 with CD5 in human and murine B and T cell lines and murine splenocytes. The interaction of CK2 holoenzyme with CD5 is mediated by the amino terminus of the regulatory subunit ␤. CK2 binds and phosphorylates CD5 at the CK2 motifs flanked by Ser 459 and Ser 461 . Cross-linking of CD5 leads to the activation of CD5-associated CK2 in a murine B-lymphoma cell line and a human T-leukemia cell line and is independent of net recruitment of CK2 to CD5. In contrast, CK2 is not activated following cross-linking of the B cell receptor complex or the T cell receptor complex. This direct regulation of CK2 by a cell surface receptor provides a novel pathway for control of cell activation that could play a significant role in regulation of CD5-dependent antigen receptor activation in T and B cells.CD5 is a 67-kDa glycoprotein that belongs to the cysteinerich macrophage scavenger receptor family of proteins expressed on all thymocytes and T cells and a subset of B cells, described as B1a B cells (CD5 B cells) (1-4). It is expressed on T cells very early in development, before the expression of the TCR-CD3 1 complex, and during progressive stages of thymocyte development, the level of CD5 expression increases, suggesting a role in thymocyte biology (5). In cells of B-lineage, the onset of CD5 expression is not well defined, but it is expressed in all Ableson transformed lines, which represent the pre-B stage (6, 7). Proposed counter-receptors for CD5 include the B cell-specific CD72, gp35-37, which is expressed on activated splenocytes and activated T cell clones, and the Ig V H framework region (8 -11). The functional significance of these candidates in context with CD5 activation has not been been established.CD5 is physically associated with the antigen receptor complex in both T and B cells and modulates intracellular signals initiated by both . The conserved cytoplasmic domain of CD5 contains four tyrosines and several sites for serine and threonine phosphorylation (15)(16)(17)(18)(19)(20). Two of the tyrosines form an imperfect immunoreceptor tyrosine activation motif (21, 22). The serine/threonine sites include four CK2-dependent serine phosphorylation sites and a protein kinase C-dependent threonine phosphorylation site. TCR crosslinking leads to rapid tyrosine phosphorylation followed by serine/threonine phosphorylation of CD5 (14,23,24). In contrast, CD5 ligation leads to tyrosine kinase activation and tyrosine phosphorylation of several substrates but only to serine phosphorylation of its own cytoplasmic domain (25,26). CD5 ...