Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.

A typical diffraction experiment will generate many images and data sets from different crystals in a very short time. This creates a challenge for the high-throughput operation of modern synchrotron beamlines as well as for the subsequent data processing. Novice users in particular may feel overwhelmed by the tables, plots and numbers that the different data-processing programs and software packages present to them. Here, some of the more common problems that a user has to deal with when processing a set of images that will finally make up a processed data set are shown, concentrating on difficulties that may often show up during the first steps along the path of turning the experiment (i.e. data collection) into a model (i.e. interpreted electron density). Difficulties such as unexpected crystal forms, issues in crystal handling and suboptimal choices of data-collection strategies can often be dealt with, or at least diagnosed, by analysing specific data characteristics during processing. In the end, one wants to distinguish problems over which one has no immediate control once the experiment is finished from problems that can be remedied a posteriori. A new software package, autoPROC, is also presented that combines third-party processing programs with new tools and an automated workflow script that is intended to provide users with both guidance and insight into the offline processing of data affected by the difficulties mentioned above, with particular emphasis on the automated treatment of multi-sweep data sets collected on multi-axis goniostats.

We present here the automated structure solution pipeline "autoSHARP." It is built around the heavy-atom refinement and phasing program SHARP, the density modification program SOLOMON, and the ARP/wARP package for automated model building and refinement (using REFMAC). It allows fully automated structure solution, from merged reflection data to an initial model, without any user intervention. We describe and discuss the preparation of the user input, the data flow through the pipeline, and the various results obtained throughout the procedure.

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused widespread outbreaks of debilitating human disease in the past five years. CHIKV invasion of susceptible cells is mediated by two viral glycoproteins, E1 and E2, which carry the main antigenic determinants and form an icosahedral shell at the virion surface. Glycoprotein E2, derived from furin cleavage of the p62 precursor into E3 and E2, is responsible for receptor binding, and E1 for membrane fusion. In the context of a concerted multidisciplinary effort to understand the biology of CHIKV, here we report the crystal structures of the precursor p62-E1 heterodimer and of the mature E3-E2-E1 glycoprotein complexes. The resulting atomic models allow the synthesis of a wealth of genetic, biochemical, immunological and electron microscopy data accumulated over the years on alphaviruses in general. This combination yields a detailed picture of the functional architecture of the 25 MDa alphavirus surface glycoprotein shell. Together with the accompanying report on the structure of the Sindbis virus E2-E1 heterodimer at acidic pH (ref. 3), this work also provides new insight into the acid-triggered conformational change on the virus particle and its inbuilt inhibition mechanism in the immature complex.

Cytochromes P450 (P450s) metabolize a wide range of endogenous compounds and xenobiotics, such as pollutants, environmental compounds, and drug molecules. The microsomal, membrane-associated, P450 isoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 are responsible for the oxidative metabolism of more than 90% of marketed drugs. Cytochrome P450 3A4 (CYP3A4) metabolizes more drug molecules than all other isoforms combined. Here we report three crystal structures of CYP3A4: unliganded, bound to the inhibitor metyrapone, and bound to the substrate progesterone. The structures revealed a surprisingly small active site, with little conformational change associated with the binding of either compound. An unexpected peripheral binding site is identified, located above a phenylalanine cluster, which may be involved in the initial recognition of substrates or allosteric effectors.

The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is the most abundant protein in the mitochondrial outer membrane (MOM). VDAC is the channel known to guide the metabolic flux across the MOM and plays a key role in mitochondrially induced apoptosis. Here, we present the 3D structure of human VDAC1, which was solved conjointly by NMR spectroscopy and x-ray crystallography. Human VDAC1 (hVDAC1) adopts a ␤-barrel architecture composed of 19 ␤-strands with an ␣-helix located horizontally midway within the pore. Bioinformatic analysis indicates that this channel architecture is common to all VDAC proteins and is adopted by the general import pore TOM40 of mammals, which is also located in the MOM.T he outer membrane of mitochondria (MOM) contains three integral membrane protein families, two of which form channels as part of larger protein complexes (for review, see ref. 1). These two MOM complexes, the general import pore TOM and the SAM insertase, allow for the entire translocation and insertion of nearly all newly synthesized proteins destined to the mitochondrial organelle (2, 3). The third protein family of typically high abundance (Ϸ10,000 copies per mitochondrion) is termed voltage-dependent anion channels (VDACs), because of the voltage sensitivity of its open probability (4, 5). Together, this small number of protein families is sufficient for full communication between mitochondria with their cellular environment (1).The VDAC channel was initially described as being reminiscent of bacterial porins and primarily responsible for the exchange of chemical energy equivalents between the cytosol and the mitochondrion (4, 6). Indeed, a variety of structural features (like barrel geometry and dimension) known from the bacterial precursors are maintained (7,8). By contrast, a variety of functions have been ascribed to the VDAC isoforms among which the direct coupling of the mitochondrial matrix to the energy maintenance of the cytosol seems to be the most general function (9). The structure of VDAC is of interest because of a substantial body of evidence connecting VDAC to apoptosis. It is suggested that VDAC is a critical player in the release of apoptogenic factors from mitochondria of mammalian cells, and consequently several hypotheses describing the mechanism of mitochondria-mediated apoptosis involving VDAC have been proposed (for review, see ref. 10). Results and DiscussionStructure Determination of hVDAC1: Combining NMR Spectroscopy and X-Ray Crystallography. In a parallel structural biology approach, we set out to characterize the structure of hVDAC1, the major isoform of this channel in mammalian tissues, by a combination of NMR spectroscopy and x-ray crystallography. The idea behind this project was to gain complementary structural information to have a solid basis for future studies, e.g., analysis of protein heterocomplex formation by NMR and crystal structures as a basis for drug target design. Only information derived from both methods and the application of an iterative s...

BUSTER±TNT is a maximum-likelihood macromolecular re®nement package. BUSTER assembles the structural model, scales observed and calculated structure-factor amplitudes and computes the model likelihood, whilst TNT handles the stereochemistry and NCS restraints/constraints and shifts the atomic coordinates, B factors and occupancies. In real space, in addition to the traditional atomic and bulk-solvent models, BUSTER models the parts of the structure for which an atomic model is not yet available (`missing structure') as lowresolution probability distributions for the random positions of the missing atoms. In reciprocal space, the BUSTER structure-factor distribution in the complex plane is a twodimensional Gaussian centred around the structure factor calculated from the atomic, bulk-solvent and missing-structure models. The errors associated with these three structural components are added to compute the overall spread of the Gaussian. When the atomic model is very incomplete, modelling of the missing structure and the consistency of the BUSTER statistical model help structure building and completion because (i) the accuracy of the overall scale factors is increased, (ii) the bias affecting atomic model re®nement is reduced by accounting for some of the scattering from the missing structure, (iii) the addition of a spatial de®nition to the source of incompleteness improves on traditional Luzzati and ' A -based error models and (iv) the program can perform selective density modi®cation in the regions of unbuilt structure alone.

Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3'-5' exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3'-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5'-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.