Structure determination of a 16.8 kDa copper protein at 2.1 a resolution using anomalous scattering data with direct methods

The structure of rusticyanin, an acid-stable copper protein, has been determined at 2.1 Å resolution by direct methods combined with the single-wavelength anomalous scattering (SAS) of copper (f″ = 3.9 e-) and then conventionally refined (Rcryst = 18.7%, Rfree = 21.9%). This is the largest unknown protein structure (Mr ≃ 16.8 kDa) to be determined using the SAS and direct-methods approach and demonstrates that by exploiting the anomalous signal at a single wavelength, direct methods can be used to determine phases at typical (∼2 Å) macromolecular crystallographic resolutions. Extrapolating from the size of the anomalous signal for copper (f″ ≃ 4 e-), this result suggests that the approach could be used for proteins with molecular weights of up to 33 kDa per Se (f″max = 8 e- at the 'white line') and 80 kDa for a Pt derivative (f″max = 19 e- at the 'white line', L3 edge). The method provides a powerful alternative in solving a de novo protein structure without either preparing multiple crystals (i.e. isomorphous heavy-atom derivative plus native crystals) or collecting multi-wavelength anomalous diffraction (MAD) data. © 1998 International Union of Crystallography all rights reserved.link_to_subscribed_fulltex

The high-resolution crystal structure of the molybdate-dependent transcriptional regulator (ModE) from Escherichia coli: a novel combination of domain folds.

The molybdate-dependent transcriptional regulator (ModE) from Escherichia coli functions as a sensor of molybdate concentration and a regulator for transcription of operons involved in the uptake and utilization of the essential element, molybdenum. We have determined the structure of ModE using multi-wavelength anomalous dispersion. Selenomethionyl and native ModE models are refined to 1. 75 and 2.1 A, respectively and describe the architecture and structural detail of a complete transcriptional regulator. ModE is a homodimer and each subunit comprises N- and C-terminal domains. The N-terminal domain carries a winged helix-turn-helix motif for binding to DNA and is primarily responsible for ModE dimerization. The C-terminal domain contains the molybdate-binding site and residues implicated in binding the oxyanion are identified. This domain is divided into sub-domains a and b which have similar folds, although the organization of secondary structure elements varies. The sub-domain fold is related to the oligomer binding-fold and similar to that of the subunits of several toxins which are involved in extensive protein-protein interactions. This suggests a role for the C-terminal domain in the formation of the ModE-protein-DNA complexes necessary to regulate transcription. Modelling of ModE interacting with DNA suggests that a large distortion of DNA is not necessary for complex formation

The evolution of synchrotron radiation and the growth of its importance in crystallography

The author's 2011 British Crystallographic Association Lonsdale Lecture included a tribute to Kathleen Lonsdale followed by detailed perspectives relevant to the title, with reference to the Synchrotron Radiation Source (SRS) and European Synchrotron Radiation Facility (ESRF). Detector initiatives have also been very important as have sample freezing cryomethods. The use of on-resonance anomalous scattering, smaller crystals, ultra-high resolution as well as the ability to handle large unit cells and the start of time-resolved structural studies have allowed a major expansion of capabilities. The reintroduction of the Laue method became a significant node point for separate development, and has also found wide application with neutron sources in biological and chemical crystallography. The UK's SRS has now been superseded by Diamond, a new synchrotron radiation source with outstanding capabilities. In Hamburg we now have access to the new ultra-low emittance PETRA III, the ultimate storage ring in effect. The ESRF Upgrade is also recently funded and takes us to sub-micrometre and even nanometre-sized X-ray beams. The very new fourth generation of the X-ray laser gives unprecedented brilliance for working with nanocrystals, and perhaps even smaller samples, such as the single molecule, with coherent X-rays, and at femtosecond time resolution

The Role of Mitochondria in the Activation/Maintenance of SOCE: Membrane Contact Sites as Signaling Hubs Sustaining Store-Operated Ca2+ Entry

Store-operated Ca2+ entry (SOCE) is a cell signaling pathway essential for immune and muscle function controlled by dynamic interactions between Ca2+-sensing STIM proteins on the endoplasmic reticulum (ER) and Ca2+-permeable ORAI channels on the plasma membrane (PM). STIM-ORAI interactions occur at membrane contact sites (MCS), evolutionarily conserved cellular structures characterized by the close apposition (10-20 nm) between the ER and target membranes that facilitate the exchange of lipids by non-vesicular transport mechanisms. STIM-ORAI interactions were considered to be restricted to ER-PM MCS, but recent evidence indicates that productive interactions take place between ER-bound STIM1 and Ca2+ channels located in intracellular organelles. Interactions between the ER and endosomes or lysosomes regulate the lipid homeostasis of these organelles and the propagation of Ca2+ signals initiated by the release of Ca2+ from acidic stores. Intracellular MCS also regulate the efficiency of phagocytosis, a fundamental cellular process essential for immunity and tissue homeostasis, by ensuring the coordinated opening of Ca2+ channels on phagocytic vacuoles and of Ca2+ release channels on juxtaposed ER stores. In this chapter, we review the current knowledge on the molecular composition and architecture of membrane contact sites that sustain Ca2+ signals at the plasma membrane and in intracellular organelles