Chemists of all fields currently publish about 50 000 crystal structures per year, the vast majority of which are X‐ray structures. We determined two molecular structures by employing electron rather than X‐ray diffraction. For this purpose, an EIGER hybrid pixel detector was fitted to a transmission electron microscope, yielding an electron diffractometer. The structure of a new methylene blue derivative was determined at 0.9 Å resolution from a crystal smaller than 1×2 μm 2 . Several thousand active pharmaceutical ingredients (APIs) are only available as submicrocrystalline powders. To illustrate the potential of electron crystallography for the pharmaceutical industry, we also determined the structure of an API from its pill. We demonstrate that electron crystallography complements X‐ray crystallography and is the technique of choice for all unsolved cases in which submicrometer‐sized crystals were the limiting factor.

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.

In two-component signal transduction, an input triggers phosphorylation of receiver domains that regulate the status of output modules. One such module is the AAA+ ATPase domain in bacterial enhancer-binding proteins that remodel the 54 form of RNA polymerase. We report X-ray solution scattering and electron microscopy structures of the activated, full-length nitrogen-regulatory protein C (NtrC) showing a novel mechanism for regulation of AAA+ ATPase assembly via the juxtaposition of the receiver domains and ATPase ring. Accompanying the hydrolysis cycle that is required for transcriptional activation, we observed major order-disorder changes in the GAFTGA loops involved in 54 binding, as well as in the DNA-binding domains. The 54 holoenzyme form of RNA polymerase forms closed DNA complexes that open only with the help of enhancer-binding proteins (EBPs). For the nitrogen-regulatory protein C of enteric bacteria (NtrC), such activity starts a cascade of events that may ultimately lead to the activation of transcription for as much as 2% of the genome (Zimmer et al. 2000). Sequence analysis of >160054 -dependent transcriptional activators shows that in addition to their oligomerization/AAA+ ATPase domain, they also have a DNA-binding domain and a regulatory domain, which in 50% of cases is a twocomponent receiver domain (Bateman et al. 2004). The helix-turn-helix DNA-binding domain recognizes enhancer-like sequences between 100 and 150 bp upstream of the promoter. In their inactive states these proteins are usually dimers that bind to pairs of tandem sites in the enhancer elements. Upon activation via the regulatory domains, they oligomerize into ATPase-active rings that use the energy from ATP hydrolysis to physically remodel closed complexes of 54 holoenzyme and promoter DNA (Rombel et al. 1998;Neuwald et al. 1999;Chaney et al. 2001;Lee et al. 2003).Crystal or NMR structures are available for several isolated domains and truncation constructs of EBPs including NtrC, DctD, PspF, ZraR, and NtrC1 (Volkman et al. 1995;Kern et al. 1999;Pelton et al. 1999;Meyer et al. 2001;Park et al. 2002;Hastings et al. 2003;Lee et al. 2003;Doucleff et al. 2005;Rappas et al. 2005;Sallai and Tucker 2005). Existing structural information on DctD and NtrC1 has been used to provide a model of how two-component signal transduction can regulate assembly of their AAA+ ATPase domains ), but this model fails to explain regulation in the closely related protein NtrC (40% sequence identity, 60% sequence similarity) Hastings et al. 2003). Here we report both SAXS/WAXS (small-and wide-angle X-ray scattering) and EM (electron microscopy) structures of the full-length, activated form of NtrC from Salmonella typhimurium. Docking of atomic models for the individual domains into the structures reveals their organization within the activated ring and uncovers a novel mechanism for the use of two-compo-

Transcription initiation by the sigma54 form of bacterial RNA polymerase requires hydrolysis of ATP by an enhancer binding protein (EBP). We present SAS-based solution structures of the ATPase domain of the EBP NtrC1 from Aquifex aeolicus in different nucleotide states. Structures of apo protein and that bound to AMPPNP or ADP-BeF(x) (ground-state mimics), ADP-AlF(x) (a transition-state mimic), or ADP (product) show substantial changes in the position of the GAFTGA loops that contact polymerase, particularly upon conversion from the apo state to the ADP-BeF(x) state, and from the ADP-AlF(x) state to the ADP state. Binding of the ATP analogs stabilizes the oligomeric form of the ATPase and its binding to sigma54, with ADP-AlF(x) having the largest effect. These data indicate that ATP binding promotes a conformational change that stabilizes complexes between EBPs and sigma54, while subsequent hydrolysis and phosphate release drive the conformational change needed to open the polymerase/promoter complex.