The aim of ARP/wARP is improved automation of model building and re®nement in macromolecular crystallography. Once a molecular-replacement solution has been obtained, it is often tedious to re®ne and rebuild the initial (search) model. ARP/wARP offers three options to automate that task to varying extents: (i) autobuilding of a completely new model based on phases calculated from the molecular-replacement solution, (ii) updating of the initial model by atom addition and deletion to obtain an improved map and (iii) docking of a structure onto a new (or mutated) sequence, followed by rebuilding and re®ning the side chains in real space. A few examples are presented where ARP/wARP made a considerable difference in the speed of structure solution and/or made possible re®nement of otherwise dif®cult or uninterpretable maps. The resolution range allowing complete autobuilding of protein structures is currently 2.0 A Ê , but for map improvement considerable advances over more conventional re®nement techniques are evident even at 3.2 A Ê spacing. Automatic refinement, building and rebuilding of models from molecularreplacement solutions.

Summary Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.

Cytotoxic T lymphocytes (CTLs) kill infected and cancerous cells. We detected transfer of cytotoxic multiprotein complexes, called supramolecular attack particles (SMAPs), from CTLs to target cells. SMAPs were rapidly released from CTLs and were autonomously cytotoxic. Mass spectrometry, immunochemical analysis, and CRISPR editing identified a carboxyl-terminal fragment of thrombospondin-1 as an unexpected SMAP component that contributed to target killing. Direct stochastic optical reconstruction microscopy resolved a cytotoxic core surrounded by a thrombospondin-1 shell of ~120 nanometer diameter. Cryo-soft x-ray tomography analysis revealed that SMAPs had a carbon-dense shell and were stored in multicore granules. We propose that SMAPs are autonomous extracellular killing entities that deliver cytotoxic cargo targeted by the specificity of shell components.

SH3 domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 domain interactions. The Grb2-like adaptor protein Mona/Gads associates stably with the T-cell receptor signal transducer SLP-76. The crystal structure of a complex between the C-terminal SH3 domain (SH3C) of Mona/Gads and a SLP-76 peptide has now been solved to 1.7 A Ê . The peptide lacks the canonical SH3 domain binding motif P±x±x±P and does not form a frequently observed poly-proline type II helix. Instead, it adopts a clamp-like shape around the circumfence of the SH3C b-barrel. The central R±x±x±K motif of the peptide forms a 3 10 helix and inserts into a negatively charged double pocket on the SH3C while several other residues complement binding through hydrophobic interactions, creating a short linear SH3C binding epitope of uniquely high af®nity. Interestingly, the SH3C displays ion-dependent dimerization in the crystal and in solution, suggesting a novel mechanism for the regulation of SH3 domain functions.