A selection of interesting papers that were published in the month before our press date in major journals most likely to report significant results in structural biology, protein and RNA folding. □ Intercellular ice propagation: experimental evidence for ice growth through membrane pores. Jason P. Acker, Janet A. Elliott, and Locksley E. McGann (2001). Biophys. J. 81, 1389–1397. cEM serves as a useful structural tool for isolated macromolecular complexes. These are easily vitrified in solution. It is more difficult to use the context of the whole cell. Vitrification is more difficult and the process of ice crystal formation is less well understood. This paper presents a study of ice crystal formation in the cellular context. Convection cryomicroscopy and video image analysis were used to examine the incidence and pattern of intracellular ice formation in the confluent monolayers of mammalian cells. It was seen that gap junctions facilitate the growth of ice crystals by allowing their propagation between cells. Low temperature and other treatments that inhibit junction formation decreased this effect. The results support the theory that gap junctions facilitate ice nucleation between cells. □ Architecture of the protein-conducting channel associated with the translating 80S ribosome. Roland Beckmann, Christian M. Spahn, Narayanan Eswar, Jürgen Helmers, Pawel A. Penczek, Andrej Sali, Joachim Frank, and Gunter Blobel (2001). Cell 107, 361–372. cEM and image reconstruction were used to determine the structure of the translating eukaryotic ribosome at 15.4 Å. Images were taken of immunopurified yeast ribosome-nascent chain complexes reconstituted with the Sec-61 complex, the protein conducting channel. The availability of the 15 Å structure of the yeast ribosome and the mapping of its components (Spahn et al. [2001], Cell 107, 373–386) allow identification of the four contacts between the 40S subunit and the channel. The binding of Sec-61 occasions a rearrangement of an rRNA expansion segment. As in previous reconstructions at lower resolution, the connection between the ribosome and the channel is not completely sealed but shows a gap of ∼15 Å. Hence the channel connection cannot serve as an ion tight seal to maintain the permeability barrier of the ER membrane. A central pore or indentation is seen in the channel and is superimposed with the exit channel for the nascent chain in the ribosome. This pore does not appear to open during translocation. The authors use their results to propose a two state model of the pore for channel function in the translocation in which the two states accommodate either secretory or membrane proteins. □ Structure of the 80S ribosome from Saccharomyces cerevisiae-tRNA-ribosome and subunit-subunit Interactions. Christian M. Spahn, Roland Beckmann, Narayanan Eswar, Pawel A. Penczek, Andrej Sali, Gunter Blobel, and Joachim Frank (2001). Cell 107, 373–386. This work employs cEM and reconstruction to determine the structure of the eukaryotic ribosome to a resolution of 15 Å. This allows a complete inventory of the eukary-otic components and a definition of the conserved and eukaryotic specific features. The authors use a threshholding method (Spahn et al. [2000], Structure Fold. Des. 8, 937–948) to separate RNA and protein density in the cEM maps. Major features of the isolated RNA density match the atomic models of the prokaryotic rRNA. This directly demonstrates the overall conservation of the rRNA components and allows the authors to map the differences including expansion segments and additional ribosomal proteins. Protein synthesis requires that the 40S and 60S subunits communicate. This is accomplished via bridges between them. Each of the seven bridges found in the bacterial 70S structure have a corresponding bridge on the 80S structure. Four additional bridges are specific to the eukaryotic structure. □ Crystal structure of a procaspase-7 zymogen: mechanisms of activation and substrate binding. Jijie Chai, Qi Wu, Eric Shiozaki, Srinivasa M. Srinivasula, Emad S. Alnemri, and Yigong Shi1 (2001). Cell 107, 399–407. Apoptosis is primarily executed by active caspases, which are derived from the inactive procaspase zymogens through proteolytic cleavage. The authors report the crystal structures of a caspase zymogen, procaspase-7, and an active caspase-7 without any bound inhibitors. Compared to the inhibitor-bound caspase-7, procaspase-7 zymogen exhibits significant structural differences surrounding the catalytic cleft, which precludes the formation of a productive conformation. Proteolytic cleavage between the large and small subunits allows rearrangement of essential loops in the active site, priming active caspase-7 for inhibitor/substrate binding. Binding by inhibitors causes a 180° flipping of the N terminus in the small subunit, which interacts with and stabilizes the catalytic cleft. These analyses reveal the structural mechanisms of caspase activation and demonstrate that the inhibitor/substrate binding is a process of induced fit. □ Structural basis for the high-affinity interaction of nidogen-1 with immunoglobulin-like domain 3 of perlecan. Marc Kvansakul, Michael Hopf, Albert Ries, Rupert Timpl, and Erhard Hohenester (2001). EMBO J. 20, 5342–5346. Nidogen and perlecan are large multifunctional basement membrane (BM) proteins conserved in all metazoa. Their high-affinity interaction, which is likely to contribute to BM assembly and function, is mediated by the central G2 domain in nidogen and the third immunoglobulin (IG)-like domain in perlecan, IG3. In the crystal structure of the mouse nidogen-1 G2–perlecan IG3 complex, perlecan IG3 belongs to the I-set of the IG superfamily and binds to the wall of the nidogen-1 G2 β-barrel. Nidogen-1 residues participating in the extensive interface are highly conserved, whereas the corresponding binding site on perlecan is more variable. □ Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-Å resolution. Stephen J. Harrop, Matthew Z. DeMaere, W. Douglas Fairlie, Tamara Reztsova, Stella M. Valenzuela, Michele Mazzanti, Raffaella Tonini, Min Ru Qiu, Lucy Jankova, Kristina Warton, Asne R. Bauskin,Wan Man Wu, Susan Pankhurst, Terence J. Campbell, Samuel N. Breit, and Paul M.G. Curmi (2001). J. Biol. Chem. 276, 44993–45000. CLIC1 is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. The crystal structure of the soluble form of CLIC1 resembles glutathione S-transferase. Glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain, with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. □ Quasi-equivalence in site-specific recombinase structure and function: crystal structure and activity of trimeric Cre recombinase bound to a three-way Lox DNA junction. Kevin C. Woods, Shelley S. Martin, Victor C. Chu, and Enoch P. Baldwin (2001). J. Mol. Biol. 313, 49–69. Doi:10.1006/jmbi.2001.5012. The crystal structure of a novel Cre-Lox synapse complex reveals a symmetric protein trimer bound to a Y-shaped three-way DNA junction, a marked departure from the typical pseudo-4-fold symmetrical tetramer. The three-way DNA junction is accommodated by a simple kink of the adjoining DNA duplexes. Adjacent Cre trimer subunits rotated 29° relative to those in the tetramers via interactions that are “quasi-equivalent” to those in the tetramer, analogous to packing differences of chemically identical viral subunits at non-equivalent positions in icosahedral capsids. □ Electrostatic stabilization of a thermophilic cold shock protein. Dieter Perl and Franz X. Schmid (2001). J. Mol. Biol. 313, 343–357. The cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus differs from its mesophilic homolog Bs-CspB from Bacillus subtilis by 15.8 kJ mol−1 in the Gibbs free energy of denaturation (GD). The two proteins vary in sequence at 12 positions but only two of them, Arg3 and Leu66 of Bc-Csp, which replace Glu3 and Glu66 of Bs-CspB, are responsible for the additional stability of Bc-Csp. These two positions are near the ends of the protein chain, but close to each other in the three-dimensional structure. The Glu3Arg exchange alone changed the stability by more than 11 kJ mol−1. Here, we elucidated the molecular origins of the stability difference between the two proteins by a mutational analysis. Electrostatic contributions to stability were characterized by measuring the thermodynamic stabili-ties of many variants as a function of salt concentration. Double and triple mutant analyses indicate that the stabilization by the Glu3Arg exchange originates from three sources. Improved hydrophobic interactions of the aliphatic moiety of Arg3 contribute about 4 kJ mol−1. Another 4 kJ mol−1 is gained from the relief of a pairwise electrostatic repulsion between Glu3 and Glu66, as in the mesophilic protein, and 3 kJ mol−1 originate from a general electrostatic stabilization by the positive charge of Arg3, which is not caused by a pairwise interaction. Mutations of all potential partners for an ion pair within a radius of 10 Å around Arg3 had only marginal effects on stability. The Glu3 Arg3 charge reversal thus optimizes ionic interactions at the protein surface by both local and global effects. However, it cannot convert the coulombic repulsion with another Glu residue into a corresponding attraction. Avoidance of unfavorable coulombic repulsions is probably a much simpler route to thermostability than the creation of stabilizing surface ion pairs, which can form only at the expense of conformational entropy. □ Conservation helps to identify biologically relevant crystal contacts. William S.J. Valdar and Janet M. Thornton (2001). J. Mol. Biol. 313, 399–416. Doi:10.1006/jmbi.2001.5034. Some crystal contacts are biologically relevant, most are not. This paper assesses the utility of combining measures of size and conservation to discriminate between biological and non-biological contacts, using a range of neural networks. For homodimers, it correctly classified 48 out of 53 biological contacts and 364 out of 366 nonbiological contacts, giving a combined accuracy of 98.3%. □ Mutational analysis of hydrogen bonding residues in the BPTI folding pathway. Grzegorz Bulaj and David P. Goldenberg (2001). J. Mol. Biol. 313, 639–656. Nine BPTI variants with replacements that remove one or more hydrogen bonds from the native protein were constructed, and the folding pathways of these proteins were determined by isolating and identifying the disulfide-bonded intermediates that accumulated during unfolding and refolding. The forward and reverse rate constants for the individual steps in the folding pathways for each protein were measured, providing a detailed description of the energetic effects of the substitutions. The native forms of eight of the nine variants were measurably destabilized, by 1–7 kcal/mol (1 cal = 4.184 J), with an average effect of 1.6 kcal/mol per hydrogen bond removed. The folding pathways for the variants were found to be similar to that previously described for the wild-type protein, with the kinetically preferred mechanism involving intramolecular rearrangements of intermediates with two disulfide bonds. Some of the substitutions, however, significantly destabilized the major intermediates and broadened the distribution of species with one or two disulfide bonds, thus identifying residues that play important roles in stabilizing the normal intermediates and defining specificity in the folding process. The kinetic data also suggest that one residue, Asn43, may play a distinctive role in defining the BPTI folding mechanism. Replacement of this residue with either Gly or Ala appeared to stabilize the major transition states for folding and unfolding. In the native protein, the side-chain of Asn43 participates directly in the hydrogen bonding pattern of the central β sheet, and the kinetic behavior of the Asn43 variants suggests that the major energy barriers in folding and unfolding may be due in part to the steric constraints imposed by this structural element, together with those imposed by the chemical transition states for thiol-disulfide exchange. □ A simple way to measure protein refolding rates in water. Daniel E. Otzen and Mikael Oliveberg (2001). J. Mol. Biol. 313, 479–483. Refolding of proteins is traditionally carried out either by diluting the denaturant-unfolded protein into buffer (GdmCl-jump) or by mixing the acid-denatured protein with strong buffer (pH-jump). The first method does not allow direct measurement of folding rates in water since the GdmCl cannot be infinitely diluted, and the second method suffers from the limitation that many proteins cannot be pH denatured. Further, some proteins do not refold reversibly from low pH where they get trapped as aggregation prone intermediates. Here, we present an alternative approach for direct measurement of refolding rates in water, which does not rely on extrapolation. The protein is denatured in SDS and is then mixed with α-cyclodextrin, which rapidly strips SDS molecules from the protein, leaving the naked unfolded protein to refold. □ The “open” and “closed” structures of the type-C inorganic pyrophosphatases from Bacillus subtilis and Streptococcus gordonii. Shinbyoung Ahn, Andrew J. Milner, Klaus Fütterer, Monika Konopka, Mohammad Ilias, Tom W. Young, and Scott A. White (2001). J. Mol. Biol. 313, 797–811. Doi: 10.1006/jmbi.2001.5070. Crystal structures of a new class of soluble inorganic pyrophosphatase (type-C PPase) from reveal a homodimeric structure with each polypeptide folding into two domains joined by a flexible hinge. The active site, formed at the interface between the N- and C-terminal domains, binds two manganese ions approximately 3.6 Å apart in a conformation resembling binuclear metal centers found in other hydrolytic enzymes. In S. gordonii PPase crystals the active site is occluded, with a sulfate ion bound in the active site. In the B. subtilis crystals the C-terminal domain is rotated by about 90°, leaving the active site wide open and accessible for substrate binding. □ β-hairpin folding simulations in atomistic detail using an implicit solvent model. Bojan Zagrovic, Eric J. Sorin, and Vijay Pande (2001). J. Mol. Biol. 313, 151–169. We have used distributed computing techniques and a supercluster of thousands of computer processors to study folding of the C-terminal β-hairpin from protein G in atomistic detail using the GB/SA implicit solvent model at 300 K. We have simulated a total of nearly 38 μs of folding time and obtained eight complete and independent folding trajectories. Starting from an extended state, we observe relaxation to an unfolded state characterized by nonspecific, temporary hydrogen bonding. This is followed by the appearance of interactions between hydrophobic residues that stabilize a bent intermediate. Final formation of the complete hydrophobic core occurs cooperatively at the same time that the final hydrogen bonding pattern appears. The folded hairpin structures we observe all contain a closely packed hydrophobic core and proper β sheet backbone dihedral angles, but they differ in backbone hydrogen bonding pattern. We show that this is consistent with the existing experimental data on the hairpin alone in solution. Our analysis also reveals short-lived semi-helical intermediates which define a thermodynamic trap. Our results are consistent with a three-state mechanism with a single rate-limiting step in which a varying final hydrogen bond pattern is apparent, and semi-helical off-pathway intermediates may appear early in the folding process. We include details of the ensemble dynamics methodology and a discussion of our achievements using this new computational device for studying dynamics at the atomic level. □ Automated data collection with a tecnai 12 electron microscope: applications for molecular imaging by cryomicroscopy. Peijun Zhang, Alexis Beatty, Jacqueline L.S. Milne, and Sriram Subramaniam (2001). J. Struct. Biol. 135, 251–261. □ Automated identification of filaments in cryoelectron microscopy images. Yuanxin Zhu, Bridget Carragher, David J. Kriegman, Ronald A. Milligan, and Clinton S. Potter (2001). J. Struct. Biol. 135, 302–312. cEM and three-dimensional image processing have made great strides in the past few years in producing higher resolution structures of macromolecular complexes. The limited resolution often reflects the poor signal to noise in the individual, low-dose images. The combination of thousands of particle images is necessary to determine structures with resolutions in the range of 7–15 Å. Higher resolution will require combining tens of thousands or more. Many workers believe the collection of large numbers of high quality micrographs remains a fundamental limitation. Automation could ease this bottleneck. These two papers provide an overview of the progress toward automating the collection of cEM data for filaments (Zhu et al.) and single particles (Zhang et al.). The work on the filaments presents a filament recognition algorithm that, in combination with the authors automated data acquisition and reconstruction system, was able to generate a reconstruction of tobacco mosaic virus to a resolution 10Å within 24 hr of first inserting the specimen. The single particle work (Zhang et al.) shows the feasibility of performing similar operations for this type of specimen with a fully computerized microscope. Both papers point out the practical limitations for automated microscopy in the determination of structures at high resolution including the performance of current CCD cameras and microscope stages. □ Analysis of Mason-Pfizer monkey virus gag particles by scanning transmission electron microscopy. Scott D. Parker, Joseph S. Wall, and Eric Hunter (2001). J. Virol. 75, 9543–9548. Retrovirus assembly generates particles with a range of sizes and shapes. This paper presents the results of STEM mass measurement for Gag particles of a type D retrovirus, Mason-Pfizer Monkey virus (MPMV). The STEM measurements indicate that the particles contain around 2000 Gag proteins. This is significantly higher than the number determined by the STEM results for Rous Sarcoma virus (RSV) virions (1500–1200) and may reflect the smaller size of the RSV particles (33 nm vs ∼55 nm) seen by cEM. The mass measurements for the MPMV particles were broadly spread (σ ≅ 20.5 MDa) confirming the variety of structures that are formed by the Gag protein during virus assembly. The paper includes a discussion of the implications of this heterogeneity for retrovirus assembly. □ Venezuelan equine encephalomyelitis virus structure and its divergence from old world alphaviruses. Angel Paredes, Kathy Alwell-Warda, Scott C. Weaver, Wah Chiu, and Stanley J. Watowich (2001). J. Virol. 75, 9532–9537. This paper presents an icosahedral image reconstruction of the enveloped alphavirus, Venezuelan Equine Encephalomyelitis virus (VEE), at 25 Å resolution from cEM. Previous reconstructions of alphaviruses have been of the Old World viruses (Semliki Forest, Sindbis, Aura, and Ross River viruses). This is the first reconstruction of an alphavirus of the New World lineage. The envelope spike glycoproteins of VEE virus have a T = 4 icosahedral arrangement and a shape similar to that seen in the previous alphavirus reconstructions. Intriguingly, the shape of the T = 4 arranged hexamers and pentamers in the nucleocapsid are distinctly different suggesting a structural difference between the two lineages. □ C terminus of infectious bursal disease virus major capsid protein vp2 is involved in definition of the t number for capsid assembly. José R. Caston, Jorge L. Martinez-Torrecuadrada, Antonio Maraver, Eleuterio Lombardo, José F. Rodriguez, J. Ignacio Casal, and José L. Carrascosa (2001). J. Virol. 75, 10815–10828. The structures of dsRNA viruses show a range of common structural features including a T = 13 shell composed of trimer clusters. The stages of organization of this shell during assembly and the factors that influence the selection of T number for many viruses remain unclear. This paper presents cEM and icosahedral image reconstruction at 28 Å of the infectious bursal disease virus (IBDV) capsids and virus-like particles formed by protein VP2 alone. When VP2 is expressed alone in insect cells, it forms dodecahedral (T = 1) particles containing 20 trimeric clusters of VP2. These may assemble further to form larger, fragile icosahedral capsids containing 12 dodecahedral particles. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and definition of the interactions between them. VP2 forms the outer protruding trimers while VP3 is found as trimers on the inner surface and may be responsible for stabilizing the capsid. Elimination of the C-terminal region of VPX is correlated with the assembly of T = 1 capsids suggesting that this domain might be involved in the induction of different conformations of VP2 during capsid morphogenesis and the direction of assembly toward the generation of the T = 13 IBDV shell. □ Crystal structure of Colicin E3: implications for cell entry and ribosome inactivation. Sandriyana Soelaiman, Karen Jakes, Nan Wu, Chunmin Li, and Menachem Shoham (2001). Mol. Cell 8, 1053–1062. Colicins kill E. coli by a process that involves binding to a surface receptor, entering the cell, and, finally, intoxicating it. The lethal action of colicin E3 is a specific cleavage in the ribosomal decoding A site. The crystal structure of colicin E3, reported here in a binary complex with its immunity protein (IP), reveals a Y-shaped molecule with the receptor binding domain forming a 100 Å long stalk and the two globular heads of the translocation domain (T) and the catalytic domain (C) comprising the two arms. Active site residues are D510, H513, E517, and R545. IP is buried between T and C. Rather than blocking the active site, IP prevents access of the active site to the ribosome. □ Structure of an archaeal homolog of the eukaryotic RNA polymerase II RPB4/RPB7 complex. Flavia Todone, Peter Brick, Finn Werner, Robert O.J. Weinzierl, and Silvia Onesti (2001). Mol. Cell 8, 1137–1143. The eukaryotic subunits RPB4 and RPB7 form a heterodimer that reversibly associates with the RNA polymerase II core and constitute the only two components of the enzyme for which no structural information is available. The authors have determined the crystal structure of the complex between the Methanococcus jannaschii subunits E and F, the archaeal homologs of RPB7 and RPB4. Subunit E has an elongated twodomain structure and contains two potential RNA binding motifs, while the smaller F subunit wraps around one side of subunit E, at the interface between the two domains. The structure supports a model for the interaction between RPB4/RPB7 and the core RNA polymerase in which the RNA binding face of RPB7 is positioned to interact with the nascent RNA transcript. □ Chemistry of ion coordination and hydration revealed by a K+ channel–Fab complex at 2.0 Å resolution. Yufeng Zhou, João H. Morais-Cabral, Amelia Kaufman, and Roderick Mackinnon (2001). Nature 414, 43–48. The structure of the KcsA K+ channel in complex with a monoclonal Fab antibody fragment at 2.0 Å resolution reveals atomic resolution features of the channel—how the K+ channel displaces water molecules around an ion at its extracellular entryway and how it holds a K+ ion in a square antiprism of water molecules in a cavity near its intracellular entryway. The selectivity filter changes its ion coordination structure in low K+ solutions. This structural change is crucial to the operation of the selectivity filter in the cellular context, where the K+ ion concentration near the selectivity filter varies in response to channel gating. □ Crystal structure of the anthrax lethal factor. Andrew D. Pannifer, Thiang Yian Wong, Robert Schwarzenbacher, Martin Renatus, Carlo Petosa, Jadwiga Bienkowska, D. Borden Lacy, R. John Collier, Sukjoon Park, Stephen H. Leppla, Philip Hanna, and Robert C. Liddington (2001). Nature 414, 229–233. Lethal factor is a 90 kDa protein that is required for anthrax pathogenesis. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase family near to their amino termini, leading to the inhibition of one or more signaling pathways. The crystal structure of LF and its complex with the N terminus of MAPKK-2 reveals a four-domain protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity. □ Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB-MoaD complex. Michael W. Lake, Margot M. Wuebbens, K.V. Rajagopalan, and Hermann Schindelin (2001). Nature 414, 325–329. The Escherichia coli proteins MoeB and MoaD are involved in a molybdenum cofactor (Moco) biosynthesis pathway that has many similarities with the E1-mediated pathway of ubiquitin activation in eukaryotes. The crystal structures of the MoeB-MoaD complex in its apo, ATP-bound, and MoaD-adenylate forms highlight the functional similarities between the MoeB- and E1-substrate complexes. These structures provide a molecular framework for understanding the activation of ubiquitin, Rub, SUMO and the sulfur incorporation step during Moco and thiamine biosynthesis. □ Crystal structure of the tricorn protease reveals a protein disassembly line. Hans Brandstetter, Jeong-Sun Kim, Michael Groll, and Robert Huber (2001). Nature 414, 466–470. The tricorn protease, is a hexameric 720K protease from Thermoplasma acidophilum that further processes the peptides generated by the proteasome. The crystal structure reveals a complex mosaic protein whereby five domains combine to form one of six subunits, which further assemble to form the 3-2-symmetric core protein. The structure shows how the individual domains coordinate the specific steps of substrate processing, including channeling of the substrate to, and the product from, the catalytic site. □ Three-dimensional reconstruction of dynamin in the constricted state. Peijun Zhang and Jenney E. Hinshaw (2001). Nat. Cell Biol. 3, 922–926. Receptor-mediated endocytosis, caveolae internalization, and some Golgi trafficking events require dynamin-dependent vesiculation. These activities require dynamin to mediate membrane constriction. This paper presents the first three-dimensional reconstruction of dynamin from cEM of tubular crystals. The reconstruction shows the constricted state at a resolution of 20 Å. The map reveals a T-shaped dimer consisting of three prominent densities: leg, stalk and head. These domains were interpreted by comparison with the known crystal structures of the dynamin PH domain and the GTPase domain of human guanylate binding protein 1. The structure suggests that the dense stalk and head regions rearrange when GTP is added to generate the force that leads to membrane constriction. □ Solution structure of a viral DNA polymerase X and evidence for a mutagenic function. Alexander K. Showalter, In-Ja L. Byeon, Mei-I Su, and Ming-Daw Tsai (2001). Nat. Struct. Biol. 8, 942–946. The African swine fever virus DNA polymerase X (ASFV Pol X or Pol X), the smallest known nucleotide polymerase, has recently been reported to be an extremely low fidelity polymerase that may be involved in strategic mutagenesis of the viral genome. The authors report the solution structure of Pol X. The structure, unique within the realm of nucleotide polymerases, consists of only palm and fingers subdomains. Despite the absence of a thumb subdomain, which is important for DNA binding in other polymerases, Pol X binds DNA with very high affinity. Further structural analyses suggest a novel mode of DNA binding that may contribute to low fidelity synthesis. We also demonstrate that the ASFV DNA ligase is a low fidelity ligase capable of sealing a nick that contains a G-G mismatch. This supports the hypothesis of a virus-encoded, mutagenic base excision repair pathway consisting of a tandem Pol X/ligase mutator. □ Structure of the Yersinia type III secretory system chaperone SycE. Sara Birtalan and Partho Ghosh (2001). Nat. Struct. Biol. 8, 974–978. In the type III secretory system of bacterial pathogens, a large number of sequence-divergent but characteristically small (14–19 kDa), acidic (pI 4–5) chaperone proteins have been identified. The authors present the 1.74 Å resolution crystal structure of the Yersinia pseudotuberculosis chaperone SycE, whose action in promoting translocation of YopE into host macrophages is essential to Yersinia pathogenesis. SycE, a compact, globular dimer with a novel fold, has two large hydrophobic surface patches that may form binding sites for YopE or other type III components. These patches are formed by structurally key residues that are conserved among many chaperones, suggesting shared structural and functional relationships. A negative electrostatic potential covers almost the entire surface of SycE and is likely conserved in character, but not in detail, among chaperones. The structure provides the first structural insights into possible modes of action of SycE and type III chaperones in general. □ Crystal structures of restrictocin-inhibitor complexes with implications for RNA recognition and base flipping. Xiaojing Yang, Tı́mea Gérczei, LaTonya Glover, and Carl C. Correll (2001). Nat. Struct. Biol. 8, 968–973. The cytotoxin sarcin disrupts elongation factor binding and protein synthesis by specifically cleaving one phosphodiester bond in ribos