Highlights

How good can our beamlines be? Liebschner D, Dauter M, Rosenbaum G, Dauter Z. Acta Crystallogr D Biol Crystallogr. 2012 Oct;68(Pt 10):1430-6.

The accuracy of X-ray diffraction data depends on the properties of the crystalline sample and on the performance of the data-collection facility (synchrotron beamline elements, goniostat, detector etc.). However, it is difficult to evaluate the level of performance of the experimental setup from the quality of data sets collected in rotation mode, as various crystal properties such as mosaicity, non-uniformity and radiation damage affect the measured intensities. A multiple-image experiment, in which several analogous diffraction frames are recorded consecutively at the same crystal orientation, allows minimization of the influence of the sample properties. A series of 100 diffraction images of a thaumatin crystal were measured on the SBC beamline 19BM at the APS (Argonne National Laboratory). The obtained data were analyzed in the context of the performance of the data-collection facility. An objective way to estimate the uncertainties of individual reflections was achieved by analyzing the behavior of reflection intensities in the series of analogous diffraction images. The multiple-image experiment is found to be a simple and adequate method to decompose the random errors from the systematic errors in the data, which helps in judging the performance of a data-collection facility. In particular, displaying the intensity as a function of the frame number allows evaluation of the stability of the beam, the beamline elements and the detector with minimal influence of the crystal properties. Such an experiment permits evaluation of the highest possible data quality potentially achievable at the particular beamline.. >> see related article
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Structural basis for prereceptor modulation of plant hormones by GH3 proteins. Westfall CS, Zubieta C, Herrmann J, Kapp U, Nanao MH, Jez JM. Science. 2012 Jun 29;336 (6089):1708-11.

Acyl acid amido synthetases of the GH3 family act as critical prereceptor modulators of plant hormone action; however, the molecular basis for their hormone selectivity is unclear. Here, we report the crystal structures of benzoate-specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid-specific AtGH3.11/JAR1. These structures, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the carboxyl-terminal domain for catalysis. We also identify residues forming the acyl acid binding site across the GH3 family and residues critical for amino acid recognition. Our results demonstrate how a highly adaptable three-dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules. >> see related article
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Structure of Apo- and Monometalated Forms of NDM-1-A Highly Potent Carbapenem-Hydrolyzing Metallo-β-Lactamase. Kim Y, Tesar C, Mire J, Jedrzejczak R, Binkowski A, Babnigg G, Sacchettini J, Joachimiak A. PLoS One. 2011;6(9):e24621. Epub 2011 Sep 8.

Structure of C3PO and mechanism of human RISC activation. Ye X, Huang N, Liu Y, Paroo Z, Huerta C, Li P, Chen S, Liu Q, Zhang H. Nat Struct Mol Biol. 2011 Jun;18(6):650-7.

The Structure of transcription factor HetR required for heterocyst differentiation in cyanobacteria. Kim Y, Joachimiak G, Ye Z, Binkowski TA, Zhang R, Gornicki P, Callahan SM, Hess WR, Haselkorn R, Joachimiak A.Proc Natl Acad Sci U S A. May 31. 2011 Jun 21;108(25):10109-14.

HetR is an essential regulator of heterocyst development in cyanobacteria. HetR binds to a DNA palindrome upstream of the hetP gene. We report the crystal structure of HetR from Fischerella at 3.0 Å. The protein is a dimer comprised of a central DNA-binding unit containing the N-terminal regions of the two subunits organized with two helix-turn-helix motifs; two globular flaps extending in opposite directions; and a hood over the central core formed from the C-terminal subdomains. The flaps and hood have no structural precedent in the protein database, therefore representing new folds. The structural assignments are supported by site-directed mutagenesis and DNA-binding studies. We suggest that HetR serves as a scaffold for assembly of transcription components critical for heterocyst development. >> see related article
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The assembly of a GTPase-kinase signalling complex by a bacterial catalytic scaffold. Selyunin AS, Sutton SE, Weigele BA, Reddick LE, Orchard RC, Bresson SM, Tomchick DR, Alto NM. Nature. 2011 Jan 6;469(7328):107-11.

The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding, and the identity of the effectors and their mechanisms of action are poorly understood. Here we identify the enterohaemorrhagic Escherichia coli O157:H7 type III effector EspG as a regulator of endomembrane trafficking using a functional screen, and report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAKs) as its relevant host substrates. The 2.5Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GTPase-activating-protein-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signalling inhibition at organelle membranes. In addition, the 2.8Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAKs are organized on adjacent surfaces of EspG, indicating its role as a 'catalytic scaffold' that effectively reprograms cellular events through the functional assembly of GTPase-kinase signalling complex. >> see related article
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Polymerization of MIP-1 chemokine (CCL3 and CCL4) and clearance of MIP-1 by insulin-degrading enzyme. Ren M, Guo Q, Guo L, Lenz M, Qian F, Koenen RR, Xu H, Schilling AB, Weber C, Ye RD, Dinner AR, Tang WJ. EMBO J. 2010 Dec 1;29(23):3952-66.

(Macrophage inflammatory protein-1 (MIP-1), MIP-1α (CCL3) and MIP-1β (CCL4) are chemokines crucial for immune responses towards infection and inflammation. Both MIP-1α and MIP-1β form high-molecular-weight aggregates. Our crystal structures reveal that MIP-1 aggregation is a polymerization process and human MIP-1α and MIP-1β form rod-shaped, double-helical polymers. Biophysical analyses and mathematical modelling show that MIP-1 reversibly forms a polydisperse distribution of rod-shaped polymers in solution. Polymerization buries receptor-binding sites of MIP-1α, thus depolymerization mutations enhance MIP-1α to arrest monocytes onto activated human endothelium. However, same depolymerization mutations render MIP-1α ineffective in mouse peritoneal cell recruitment. Mathematical modelling reveals that, for a long-range chemotaxis of MIP-1, polymerization could protect MIP-1 from proteases that selectively degrade monomeric MIP-1. Insulin-degrading enzyme (IDE) is identified as such a protease and decreased expression of IDE leads to elevated MIP-1 levels in microglial cells. Our structural and proteomic studies offer a molecular basis for selective degradation of MIP-1. The regulated MIP-1 polymerization and selective inactivation of MIP-1 monomers by IDE could aid in controlling the MIP-1 chemotactic gradient for immune surveillance.>> see related article
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Munc13 C2B domain is an activity-dependent Ca2+ regulator of synaptic exocytosis. Shin OH, Lu J, Rhee JS, Tomchick DR, Pang ZP, Wojcik SM, Camacho-Perez M, Brose N, Machius M, Rizo J, Rosenmund C, Südhof TC. Nat Struct Mol Biol. 2010 Mar;17(3):280-8.

Munc13 is a multidomain protein present in presynaptic active zones that mediates the priming and plasticity of synaptic vesicle exocytosis, but the mechanisms involved remain unclear. Here we use biophysical, biochemical and electrophysiological approaches to show that the central C(2)B domain of Munc13 functions as a Ca(2+) regulator of short-term synaptic plasticity. The crystal structure of the C(2)B domain revealed an unusual Ca(2+)-binding site with an amphipathic alpha-helix. This configuration confers onto the C(2)B domain unique Ca(2+)-dependent phospholipid-binding properties that favor phosphatidylinositolphosphates. A mutation that inactivated Ca(2+)-dependent phospholipid binding to the C(2)B domain did not alter neurotransmitter release evoked by isolated action potentials, but it did depress release evoked by action-potential trains. In contrast, a mutation that increased Ca(2+)-dependent phosphatidylinositolbisphosphate binding to the C(2)B domain enhanced release evoked by isolated action potentials and by action-potential trains. Our data suggest that, during repeated action potentials, Ca(2+) and phosphatidylinositolphosphate binding to the Munc13 C(2)B domain potentiate synaptic vesicle exocytosis, thereby offsetting synaptic depression induced by vesicle depletion..>> see related article
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Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35. Leung DW, Prins KC, Borek DM, Farahbakhsh M, Tufariello JM, Ramanan P, Nix JC, Helgeson LA, Otwinowski Z, Honzatko RB, Basler CF, Amarasinghe GK. Nat Struct Mol Biol. 2010 Feb;17(2):165-72.

Ebola viral protein 35 (VP35), encoded by the highly pathogenic Ebola virus, facilitates host immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I-like receptors. Here we report the crystal structure of the Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which together with in vivo results reveals how VP35-dsRNA interactions contribute to immune evasion. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are 'end-capped' by a pocket of hydrophobic residues that mimic RIG-I-like receptor recognition of blunt-end dsRNA. Residues critical for RNA binding are also important for interferon inhibition in vivo but not for viral polymerase cofactor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.>> see related article
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Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor. Wu K, Li W, Peng G, Li F. Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19970-4.

NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). We investigated how different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors. We determined the crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2. NL63-CoV RBD has a novel beta-sandwich core structure consisting of 2 layers of beta-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homology in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a "virus-binding hotspot" on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.>> see related article
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Structural basis for the inhibition of human 5,10-methenyltetrahydrofolate synthetase by N10-substituted folate analogues. Wu D, L Y, Song G, Cheng C, Zhang R, Joachimiak A, Shaw N, Liu ZJ. Cancer Res. 2009 Sep 15;69(18):7294-301.

5,10-Methenyltetrahydrofolate synthetase (MTHFS) regulates the flow of carbon through the one-carbon metabolic network, which supplies essential components for the growth and proliferation of cells. Inhibition of MTHFS in human MCF-7 breast cancer cells has been shown to arrest the growth of cells. Absence of the three-dimensional structure of human MTHFS (hMTHFS) has hampered the rational design and optimization of drug candidates. Here, we report the structures of native hMTHFS, a binary complex of hMTHFS with ADP, hMTHFS bound with the N5-iminium phosphate reaction intermediate, and an enzyme-product complex of hMTHFS. The N5-iminium phosphate captured for the first time in our crystal structure unravels a unique strategy used by hMTHFS for recognition of the substrate and provides structural basis for the regulation of enzyme activity. Binding of N10-substituted folate analogues places Y152 in the middle of the channel connecting ATP binding site with the substrate binding pocket, precluding the positioning of gamma-phosphate for a nucleophilic attack. Using the structures of hMTHFS as a guide, we have probed the role of residues surrounding the active site in catalysis by mutagenesis. The ensemble of hMTHFS structures and the mutagenesis data yield a coherent picture of the MTHFS active site, determinants of substrate specificity, and new insights into the mechanism of inhibition of hMTHFS.>> see related article
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Crystal structure of Bacillus anthracis transpeptidase enzyme CapD.
Wu R, Richter S, Zhang RG, Anderson VJ, Missiakas D, Joachimiak A.
The rBacillus anthracis elaborates a poly-gamma-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the gamma-glutamyltranspeptidase CapD with and without alpha-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-gamma-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-gamma-glutamate binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro(427), Gly(428), and Gly(429) activate the catalytic residue of the enzyme, Thr(352), and stabilize an oxyanion hole via main chain amide hydrogen bonds.>> see related article
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X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus.
Bornholdt ZA, Prasad BV
The recent emergence of highly pathogenic avian (H5N1) influenza viruses, their epizootic and panzootic nature, and their association with lethal human infections have raised significant global health concerns. Several studies have underlined the importance of non-structural protein NS1 in the increased pathogenicity and virulence of these strains. NS1, which consists of two domains-a double-stranded RNA (dsRNA) binding domain and the effector domain, separated through a linker-is an antagonist of antiviral type-I interferon response in the host. Here we report the X-ray structure of the full-length NS1 from an H5N1 strain (A/Vietnam/1203/2004) that was associated with 60% of human deaths in an outbreak in Vietnam. Compared to the individually determined structures of the RNA binding domain and the effector domain from non-H5N1 strains, the RNA binding domain within H5N1 NS1 exhibits modest structural changes, while the H5N1 effector domain shows significant alteration, particularly in the dimeric interface. Although both domains in the full-length NS1 individually participate in dimeric interactions, an unexpected finding is that these interactions result in the formation of a chain of NS1 molecules instead of distinct dimeric units. Three such chains in the crystal interact with one another extensively to form a tubular organization of similar dimensions to that observed in the cryo-electron microscopy images of NS1 in the presence of dsRNA. The tubular oligomeric organization of NS1, in which residues implicated in dsRNA binding face a 20-A-wide central tunnel, provides a plausible mechanism for how NS1 sequesters varying lengths of dsRNA, to counter cellular antiviral dsRNA response pathways, while simultaneously interacting with other cellular ligands during an infection.>> see related article
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Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA.
Yang CG, Yi C, Duguid EM, Sullivan CT, Jian X, Rice PA, He C.

Escherichia coli AlkB and its human homologues ABH2 and ABH3 repair DNA/RNA base lesions by using a direct oxidative dealkylation mechanism. ABH2 has the primary role of guarding mammalian genomes against 1-meA damage by repairing this lesion in double-stranded DNA (dsDNA), whereas AlkB and ABH3 preferentially repair single-stranded DNA (ssDNA) lesions and can repair damaged bases in RNA. Here we show the first crystal structures of AlkB-dsDNA and ABH2-dsDNA complexes, stabilized by a chemical cross-linking strategy. This study reveals that AlkB uses an unprecedented base-flipping mechanism to access the damaged base: it squeezes together the two bases flanking the flipped-out one to maintain the base stack, explaining the preference of AlkB for repairing ssDNA lesions over dsDNA ones. In addition, the first crystal structure of ABH2, presented here, provides a structural basis for designing inhibitors of this human DNA repair protein.>> see related article
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Structural Dynamics of α-Actinin-Vinculin Interactions
Philippe R. J. Bois, Robert A. Borgon, Clemens Vonrhein, and Tina Izard

α-Actinin activates vinculin in adhesion complexes. The α-actinin dimer (red and green) binds to vinculin through the agency of a vinculin binding site present at the end of its rod domain, which is proposed to swing out to bind to vinculin. α-Actinin binds the N-terminal helical bundle of vinculin (pink), disrupting its interactions with vinculin's tail domain (cyan)>> see related article
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Crystal structure of the CorA Mg2+ transporter Vladimir V. Lunin, Elena Dobrovetsky, Galina Khutoreskaya , Rongguang Zhang, Andrzej Joachimiak, Declan A. Doyle, Alexey Bochkarev, Michael E. Maguire, Aled M. Edwards and Christopher M. Koth The magnesium ion, Mg2+, is essential for myriad biochemical processes and remains the only major biological ion whose transport mechanisms remain unknown. The CorA family of magnesium transporters is the primary Mg2+ uptake system of most prokaryotes and a functional homologue of the eukaryotic mitochondrial magnesium transporter. Here we determine crystal structures of the full-length Thermotoga maritima CorA in an apparent closed state and its isolated cytoplasmic domain at 3.9 Å and 1.85 Å resolution, respectively. The transporter is a funnel-shaped homopentamer with two transmembrane helices per monomer.>> see related article

........................................................................................................................................................ Structure of tracheal cytotoxin in complex with a heterodimeric pattern-recognition receptor. Chung-I Chang1,2, Yogarany Chelliah 1,2, Dominika Borek 2, Dominique Mengin-Lecreulx 3 and Johann Deisenhofer 1,2 Tracheal cytotoxin (TCT), a naturally occurring fragment of Gram-negative peptidoglycan, is a potent elicitor of innate immune responses in Drosophila. It induces the heterodimerization of its recognition receptors, the peptidoglycan recognition proteins (PGRPs) LCa and LCx, which activates the immune deficiency pathway... >> see related article ....................................................................................................................................................... Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism. Nature 443, 870-874 (19 October 2006) Yuequan Shen 1, Andrzej Joachimiak 2, Marsha Rich Rosner 1 and Wei-Jen Tang1 Insulin-degrading enzyme (IDE), a Zn2+-metalloprotease, is involved in the clearance of insulin and amyloid-beta (refs 1–3). Loss-of-function mutations of IDE in rodents cause glucose intolerance and cerebral accumulation of amyloid-beta, whereas enhanced IDE activity effectively reduces brain amyloid-beta (refs 4–7). Here we report structures of human IDE in complex with four substrates (insulin B chain, amyloid-beta peptide (1–40), amylin and glucagon). The amino- and carboxy-terminal domains of IDE (IDE-N and IDE-C, respectively) form an enclosed cage just large enough to encapsulate insulin. >> see related article ....................................................................................................................................................... A Bird Flu Protein Link to Virulence Smart viruses infect without killing the host, at least in the short term. In such low-level infections, the virus gains time to persist, reproduce, infect other individuals, and spread through the population. Some viruses persist in certain host populations without killing them, but in other hosts they have a rapid and deadly effect. The H5N1 virus strains responsible for recent lethal outbreaks of bird flu apparently existed for quite some time in wild goose populations without doing much harm. >> see related article ....................................................................................................................................................... Ultrahigh-resolution study of protein atomic displacement parameters at cryotemperatures obtained with a helium cryostat Tatiana Petrova,a,b,c Stephan Ginell,aAndre Mitschler,c Isabelle Hazemann,c Thomas Schneider,d Alexandra Cousido,c Vladimir Y. Lunin,b Andrzej Joachimiak a* and Alberto Podjarnyc Two X-ray data sets for a complex of human aldose reductase (h-AR) with the inhibitor IDD 594 and the cofactor NADP+ were collected from two different parts of the same crystal to a resolution of 0.81 Å at 15 and 60 K using cold helium gas as cryogen. The contribution of temperature to the atomic B values was estimated by comparison of the independently refined models. It was found that although being slightly different for different kinds of atoms, the differences (B) in the isotropic equivalents B of atomic displacement parameters (ADPs) were approximately constant (about 1.7 Å2) for well ordered atoms as the temperature was increased from 15 to 60 K ...>> see related article ................................................................................................................................................... The transition state for formation of the peptide bond in the ribosome Asta Gindulyte*, Anat Bashan†, Ilana Agmon†, Lou Massa*, Ada Yonath†‡, and Jerome Karle§ >> Proc Natl Acad Sci U S A. 2006 Sep 5;103(36):13327-32 Using quantum mechanics and exploiting known crystallographic coordinates of tRNA substrate located in the ribosome peptidyl transferase center around the 2-fold axis, we have investigated the mechanism for peptide-bond formation. The calculation is based on a choice of 50 atoms assumed to be important in the mechanism. We used density functional theory to optimize the geometry and energy of the transition state (TS) for peptide-bond formation. The TS is formed simultaneously with the rotatory motion enabling the translocation of the A-site tRNA 3' end into the P site, and we estimated the magnitude of rotation angle between the A-site starting position and the place at which the TS occurs >> see related article ....................................................................................................................................................... The many faces of radiation-induced changes Dominika Borek,a Stephan L. Ginell,b Marcin Cymborowski,c Wladek Minorc and Zbyszek Otwinowski a* >> J. Synchrotron Rad. (2007) 14, 24-33 During diffraction experiments even cryo-cooled protein crystals can be significantly damaged due to chemical and physical changes induced by absorbed X-ray photons. The character and scale of the observed effects depend strongly on the temperature and the composition of crystals. The absorption of radiation energy results in incremental regular changes to the crystal structure, making its impact on the process of solving the structure strongly correlated with other experimental variables. An understanding of all the dependencies is still limited and does not allow for a precise prediction of the outcome of a particular diffraction experiment. Results are presented of diffraction experiments performed under different experimental conditions. The influence of temperature and crystal composition on different characteristics of radiation damage is analyzed. The observed effects are discussed in terms of their impact on data processing and phasing procedures. >> see related article ....................................................................................................................................................... Crystal structure of poliovirus 3CD protein: virally encoded protease and precursor to the RNA-dependent RNA polymerase Laura L. Marcotte,1 Amanda B. Wass,1 David W. Gohara,1,Harsh B. Pathak,2, Jamie J. Arnold,2 David J. Filman,2 Craig E. Cameron,2 and James M. Hogle1 J. Virol. 81 (7), April, 3583-3596 (2007) Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3C (pro) and the viral polymerase 3D(pol) and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-A resolution and the G64S fidelity mutant of 3D(pol)at a 3.0-A resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. >> see related article ....................................................................................................................................................... Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function Olga Koroleva1, Nodar Makharashvili1, Charmain T Courcelle2, Justin Courcelle2 and Sergey Korolev1 The EMBO Journal (2007) 26, 867–877 RecF, together with RecO and RecR, belongs to a ubiquitous group of recombination mediators (RMs) that includes eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA-like recombinases and displacing single-stranded DNA-binding proteins. Here, we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled-coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so-called 'Lobe II' subdomain of Rad50... >> see related article   ....................................................................................................................................................... Domain Architecture of Pyruvate Carboxylase, a Biotin-Dependent Multifunctional Enzyme Martin St. Maurice,1 Laurie Reinhardt,1 Kathy H. Surinya,2 Paul V. Attwood,3 John C. Wallace,2 W. Wallace Cleland,1 Ivan Rayment1 Science 24 August 2007: Vol. 317. no. 5841, pp. 1076 - 1079 Biotin-dependent multifunctional enzymes carry out metabolically important carboxyl group transfer reactions and are potential targets for the treatment of obesity and type 2 diabetes. These enzymes use a tethered biotin cofactor to carry an activated carboxyl group between distantly spaced active sites. The mechanism of this transfer has remained poorly understood. Here we report the complete structure of pyruvate carboxylase at 2.0 angstroms resolution, which shows its domain arrangement. The structure, when combined with mutagenic analysis, shows that intermediate transfer occurs between active sites on separate polypeptide chains. >> see related article ....................................................................................................................................................... Structure and Function of an Essential Component of the Outer Membrane Protein Assembly Machine Seokhee Kim,1 Juliana C. Malinverni,2 Piotr Sliz,3,4 Thomas J. Silhavy,2 Stephen C. Harrison,3,4 Daniel Kahne1,3 Science 17 August 2007: Vol. 317. no. 5840, pp. 961 - 964 Integral ß-barrel proteins are found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. The machine that assembles these proteins contains an integral membrane protein, called YaeT in Escherichia coli, which has one or more polypeptide transport–associated (POTRA) domains. The crystal structure of a periplasmic fragment of YaeT reveals the POTRA domain fold and suggests a model for how POTRA domains can bind different peptide sequences, as required for a machine that handles numerous ß-barrel protein precursors. Analysis of POTRA domain deletions shows which are essential and provides a view of the spatial organization of this assembly machine.>> see related article ....................................................................................................................................................... Synthetic antibodies for specific recognition and crystallization of structured RNA. Ye JD, Tereshko V, Frederiksen JK, Koide A, Fellouse FA, Sidhu SS, Koide S, Kossiakoff AA, Piccirilli JA. Proc Natl Acad Sci U S A. 2008 Jan 8;105(1):82-7 Antibodies that bind protein antigens are indispensable in biochemical research and modern medicine. However, knowledge of RNA-binding antibodies and their application in the ever-growing RNA field is lacking. Here we have developed a robust approach using a synthetic phage-display library to select specific antigen-binding fragments (Fabs) targeting a large functional RNA. We have solved the crystal structure of the first Fab-RNA complex at 1.95 Å. Capability in phasing and crystal contact formation suggests that the Fab provides a potentially valuable crystal chaperone for RNA. The crystal structure reveals that the Fab achieves specific RNA binding on a shallow surface with complementarity-determining region (CDR) sequence diversity, length variability, and main-chain conformational plasticity. The Fab-RNA interface also differs significantly from Fab-protein interfaces in amino acid composition and light-chain participation. These findings yield valuable insights for engineering of Fabs as RNA-binding modules and facilitate further development of Fabs as possible therapeutic drugs and biochemical tools to explore RNA biology. >> see related article ....................................................................................................................................................... Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase Matthew P. Blakeley, Federico Ruiz, Raul Cachau, Isabelle Hazemann, Flora Meilleur, Andre Mitschler, Stephan Ginell, Pavel Afonine, Oscar N. Ventura, Alexandra Cousido-Siah, Michael Haertlein, Andrzej Joachimiak, Dean Myles, and Alberto Podjarny PNAS | February 12, 2008 vol. 105 | no. 6 | 1844-1848 Enzymatic catalysis frequently involves proton translocation (PT). PT can occur on very short time scales (nanoseconds) and over long distances (tens of Angstroms), often involving multiple proton relay sites and proton wires (Grotthus-like mechanisms) (1). The nature of PT processes combined with the weak x-ray scattering signal from hydrogen atoms, which excludes their observation at resolutions <1.2 Å, make the structural characterization of PT processes very difficult. Recent advances in synchrotron and neutron sources, detectors, cryocooling, and software, coupled with the ability to obtain high-quality crystals, have led to an improved level of detail in protein structures, as exemplified in our recent structure of human aldose reductase (h-AR) determined at subatomic resolution (2) >> see related article   NADP Regulates the Yeast GAL Induction System P. Rajesh Kumar, Yao Yu, Rolf Sternglanz, Stephen Albert Johnston, Leemor Joshua-Tor Science 22 February 2008: Vol. 319. no. 5866, pp. 1090 - 1092 Transcriptional regulation of the galactose-metabolizing genes in Saccharomyces cerevisiae depends on three core proteins: Gal4p, the transcriptional activator that binds to upstream activating DNA sequences (UASGAL); Gal80p, a repressor that binds to the carboxyl terminus of Gal4p and inhibits transcription; and Gal3p, a cytoplasmic transducer that, upon binding galactose and adenosine 5'-triphosphate, relieves Gal80p repression. The current model of induction relies on Gal3p sequestering Gal80p in the cytoplasm. However, the rapid induction of this system implies that there is a missing factor. Our structure of Gal80p in complex with a peptide from the carboxyl-terminal activation domain of Gal4p reveals the existence of a dinucleotide that mediates the interaction between the two. Biochemical and in vivo experiments suggests that nicotinamide adenine dinucleotide phosphate (NADP) plays a key role in the initial induction event.>> see related article