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1.  Structures of monomeric, dimeric and trimeric PCNA: PCNA-ring assembly and opening 
Crystal structures of three PCNA homologues from Sulfolobus solfataricus have been determined in different oligomeric states. The structural features in different assemblies present a molecular model for clamp-ring forming and opening.
DNA sliding clamps form an oligomeric ring encircling DNA and serve as a moving platform for DNA-processing proteins. The opening and closing of a sliding-clamp ring is essential to load the clamp onto DNA in order to perform its functions. The molecular details of how clamp rings open and enclose DNA are still not clear. Three PCNA homologues have been found in Sulfolobus solfataricus which form a heterotrimer. Taking advantage of their hetero-oligomeric nature, the structures of the PCNAs in monomeric PCNA3, dimeric PCNA1–PCNA2 and trimeric PCNA1–PCNA2–PCNA3 forms were determined at resolutions of 2.6–1.9 Å. The distinct oligomeric structures represent different stages in ring formation, which were verified in solution by ultra­centrifugation analysis. The heterodimer opens in a V-shape of 130°, while the heterotrimers form a ring with a 120° rotation between monomers. The association of a rigid PCNA3 monomer with an opened PCNA1–PCNA2 heterodimer closes the ring and introduces a spring tension in the PCNA1–PCNA2 interface, thus bending the nine-stranded intermolecular β-sheet to fit the 120° rotation. The release of the spring tension as PCNA3 dissociates from the ring may facilitate ring opening. The structural features in different assemblies present a molecular model for clamp ring assembly and opening.
doi:10.1107/S0907444908021665
PMCID: PMC3606083  PMID: 18703842
DNA replication; DNA sliding clamps; PCNA-ring opening; conformational transitions
2.  Crystal structure of an isolated, unglycosylated antibody CH2 domain 
The CH2 (CH3 for IgM and IgE) domain of an antibody plays an important role in mediating effector functions and preserving antibody stability. It is the only domain in human immunoglobulins (Igs) which is involved in weak interchain protein-protein interactions with another CH2 domain solely through sugar moieties. The N-linked glycosylation at Asn297 is conserved for mammalian IgGs as well as homologous regions of other antibody isotypes. To examine the structural details of the CH2 domain in the absence of glycosylation and other antibody domains, we determined the crystal structure of an isolated, unglycosylated antibody γ1 CH2 domain at 1.7 Å, and compared it with the corresponding CH2 structures from intact Fc, IgG and Fc receptor complexes. Furthermore, we studied the oligomeric state of the protein in solution using size exclusion chromatography. The results suggested that the unglycosylated human antibody CH2 domain is a monomer and its structure is similar to that found in the intact Fc, IgG and Fc receptor complex structures. However, we observed certain structural variations along the Fc receptor binding sites. Owing to the small size, stability and non-immunogenic Ig template, the CH2 domain structure could be useful for the development of antibody domains exerting some effector functions and/or antigen specificity if made by protein design, and, as a robust scaffold in protein engineering applications.
doi:10.1107/S0907444908025274
PMCID: PMC2596763  PMID: 18931413
antibody; immunoglobulin; unglycosylated CH2, IgG; Fc; CH2 domain
3.  Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain 
The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance.
Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinities on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn2+-bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn2+ ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn2+ binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels.
doi:10.1107/S0907444908023512
PMCID: PMC2725782  PMID: 18931410
CRISPs; CNG ion-channel blockers; snake venoms; Zn2+ binding; cysteine-rich domain
4.  Towards a rational approach for heavy-atom derivative screening in protein crystallography 
Heavy-atom derivatization is routinely used in protein structure determination and is thus of critical importance in structural biology. In order to replace the current trial-and-error heavy-atom derivative screening with a knowledge-based rational derivative-selection method, the reactivity of more than 40 heavy-atom compounds over a wide range of buffer and pH values was systematically examined using peptides which contained a single reactive amino-acid residue.
Heavy-atom derivatization is routinely used in protein structure determination and is thus of critical importance in structural biology. In order to replace the current trial-and-error heavy-atom derivative screening with a knowledge-based rational derivative-selection method, the reactivity of more than 40 heavy-atom compounds over a wide range of buffer and pH values was systematically examined using peptides which contained a single reactive amino-acid residue. Met-, Cys- and His-containing peptides were derivatized against Hg, Au and Pt compounds, while Tyr-, Glu-, Asp-, Asn- and Gln-containing peptides were assessed against Pb compounds. A total of 1668 reactive conditions were examined using mass spectrometry and were compiled into heavy-atom reactivity tables (http://sis.niaid.nih.gov/cgi-bin/heavyatom_reactivity.cgi). The results showed that heavy-atom derivatization reactions are highly linked to buffer and pH, with the most accommodating buffer being MES at pH 6. A group of 21 compounds were identified as most successful irrespective of ligand or buffer/pH conditions. To assess the applicability of the peptide heavy-atom reactivity to proteins, lysozyme crystals were derivatized with a list of peptide-reactive compounds that included both known and new compounds for lysozyme derivatization. The results showed highly consistent heavy-atom reactivities between the peptides and lysozyme.
doi:10.1107/S0907444907068849
PMCID: PMC2725783  PMID: 18391402
heavy-atom derivatization; heavy-atom screening
5.  Structural Analysis of a Holo Enzyme Complex of Mouse Dihydrofolate Reductase with NADPH and a Ternary Complex with the Potent and Selective Inhibitor, 2,4-Diamino-6-(2′-hydroxydibenz[b,f]azepin-5-yl)methylpteridine 
It has been shown that 2,4-diamino-6-arylmethylpteridines and 2,4-diamino-5-arylmethylpyrimidines containing a O-carboxylalkyloxy group in the aryl moiety are potent and selective inhibitors of the dihydrofolate reductase (DHFR) from such opportunistic pathogens as Pneumocystis carinii, the causative agent of Pneumocystis pneumonia in HIV AIDS patients. In order to understand the structure-activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structures of mouse (m) DHFR, a mammalian homologue, holo and ternary complexes with NADPH and the inhibitor 2,4-diamino-6-(2′-hydroxydibenz[b,f]azepin-5-yl)methylpteridine were determined to 1.9Ǻ and 1.4Ǻ resolution, respectively. Structural data for the ternary complex with the potent O-(3-carboxypropyl) inhibitor PT684 revealed no electron density for the O-carboxylalkyloxy side chain. The side chain either was cleaved or was completely disordered. The electron density fit the less potent hydroxyl compound, PT684a. Additionally, co-crystallization of mDHFR with NADPH and the less potent 2′-(4-carboxybenzyl) inhibitor PT682 showed no electron density for the inhibitor and resulted in the first report of a holo enzyme complex despite several attempts at crystallization of a ternary complex. Modeling data of PT682 in the active site of mDHFR and pcDHFR indicate binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit in the active site or that the inhibitor side chain would have to adopt an alternative binding mode than that observed for other carboxyalkyloxy inhibitors. These data also show that the mDHFR complexes have a decreased active site volume as reflected in the relative shift of helix C (residues 59-64) by 0.6Ǻ, compared to Pneumocystis carinii DHFR ternary complexes. These data are consistent with the greater inhibitory potency against pcDHFR.
doi:10.1107/S0907444908022348
PMCID: PMC2615397  PMID: 18703847
6.  Structure of the thioredoxin-like domain of yeast glutaredoxin 3 
Yeast glutaredoxin 3 (Grx3) is a cytosolic protein that regulates the activity of the iron-responsive transcriptional activator Aft1. This member of the monothiol glutaredoxin family contains a thioredoxin-like domain and a glutaredoxin-like domain, which both possess a monothiol active site. The crystal structure of the thioredoxin-like domain has been determined at 1.5 Å resolution and represents the first published structure of this domain for the monothiol glutaredoxin family. The loop containing the signature motif WAxxC is partially disordered, indicating a greater degree of flexibility in this region compared with classical dithiol thioredoxins with a WCGPC active-site motif.
doi:10.1107/S0907444908021641
PMCID: PMC2581515  PMID: 18703840
7.  Structure of an isolated unglycosylated antibody CH2 domain 
The crystal structure of an isolated unglycosylated antibody CH2 domain has been determined at 1.7 Å resolution.
The CH2 (CH3 for IgM and IgE) domain of an antibody plays an important role in mediating effector functions and preserving antibody stability. It is the only domain in human immunoglobulins (Igs) which is involved in weak interchain protein–protein interactions with another CH2 domain solely through sugar moieties. The N-linked glycosylation at Asn297 is conserved in mammalian IgGs as well as in homologous regions of other antibody isotypes. To examine the structural details of the CH2 domain in the absence of glycosylation and other antibody domains, the crystal structure of an isolated unglycosylated antibody γ1 CH2 domain was determined at 1.7 Å resolution and compared with corresponding CH2 structures from intact Fc, IgG and Fc receptor complexes. Furthermore, the oligomeric state of the protein in solution was studied using size-exclusion chromatography. The results suggested that the unglycosylated human antibody CH2 domain is a monomer and that its structure is similar to that found in the intact Fc, IgG and Fc receptor complex structures. However, certain structural variations were observed in the Fc receptor-binding sites. Owing to its small size, stability and non-immunogenic Ig template, the CH2-domain structure could be useful for the development by protein design of antibody domains exerting effector functions and/or antigen specificity and as a robust scaffold in protein-engineering applications.
doi:10.1107/S0907444908025274
PMCID: PMC2596763  PMID: 18931413
antibodies; immunoglobulins; unglycosylated CH2; IgG; Fc; CH2 domains
8.  High-molecular-weight polymers for protein crystallization: poly-γ-glutamic acid-based precipitants 
High-molecular-weight poly-γ-glutamic acid-based polymers have been synthesized, tested and adopted for protein crystallization.
Protein crystallization has been revolutionized by the intro­duction of high-throughput technologies, which have led to a speeding up of the process while simultaneously reducing the amount of protein sample necessary. Nonetheless, the chemistry dimension of protein crystallization has remained relatively undeveloped. Most crystallization screens are based on the same set of precipitants. To address this shortcoming, the development of new protein precipitants based on poly-γ-­glutamic acid (PGA) polymers with different molecular-weight ranges is reported here: PGA-LM (low molecular weight) of ∼400 kDa and PGA-HM (high molecular weight) of >1000 kDa. It is also demonstrated that protein precipitants can be expanded further to polymers with much higher molecular weight than those that are currently in use. Furthermore, the modification of PGA-like polymers by covalent attachments of glucosamine substantially improved their solubility without affecting their crystallization properties. Some preliminary PGA-based screens are presented here.
doi:10.1107/S0907444908021616
PMCID: PMC2526455  PMID: 18703844
protein crystallization; crystallization precipitants; crystallization screens; high throughput
9.  Structure of the thioredoxin-like domain of yeast glutaredoxin 3 
Yeast Grx3 is involved in iron-responsive transcription regulation. The single active site thiol of the thioredoxin-like domain is in a flexible surface loop as suggested by its partial disorder.
Yeast glutaredoxin 3 (Grx3) is a cytosolic protein that regulates the activity of the iron-responsive transcriptional activator Aft1. This member of the monothiol glutaredoxin family contains a thioredoxin-like domain and a glutaredoxin-like domain, which both possess a monothiol active site. The crystal structure of the thioredoxin-like domain has been determined at 1.5 Å resolution and represents the first published structure of this domain for the monothiol glutaredoxin family. The loop containing the signature motif WAxxC is partially disordered, indicating a greater degree of flexibility in this region compared with classical dithiol thioredoxins with a WCGPC active-site motif.
doi:10.1107/S0907444908021641
PMCID: PMC2581515  PMID: 18703840
glutaredoxin 3; thioredoxin-like domain
10.  Structural analysis of a holoenzyme complex of mouse dihydrofolate reductase with NADPH and a ternary complex with the potent and selective inhibitor 2,4-diamino-6-(2′-hydroxydibenz[b,f]azepin-5-yl)methylpteridine 
The structures of mouse DHFR holo enzyme and a ternary complex with NADPH and a potent inhibitor are described.
It has been shown that 2,4-diamino-6-arylmethylpteridines and 2,4-diamino-5-arylmethylpyrimidines containing an O-carboxylalkyloxy group in the aryl moiety are potent and selective inhibitors of the dihydrofolate reductase (DHFR) from opportunistic pathogens such as Pneumocystis carinii, the causative agent of Pneumocystis pneumonia in HIV/AIDS patients. In order to understand the structure–activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structures of mouse DHFR (mDHFR; a mammalian homologue) holo and ternary complexes with NADPH and the inhibitor 2,4-diamino-6-(2′-hydroxy­dibenz[b,f]azepin-5-yl)methylpteridine were determined to 1.9 and 1.4 Å resolution, respectively. Structural data for the ternary complex with the potent O-(3-carboxypropyl) inhibitor PT684 revealed no electron density for the O-carboxylalkyloxy side chain. The side chain was either cleaved or completely disordered. The electron density fitted the less potent hydroxyl compound PT684a. Additionally, cocrystallization of mDHFR with NADPH and the less potent 2′-(4-carboxybenzyl) inhibitor PT682 showed no electron density for the inhibitor and resulted in the first report of a holoenzyme complex despite several attempts at crystallization of a ternary complex. Modeling data of PT682 in the active site of mDHFR and P. carinii DHFR (pcDHFR) indicate that binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit into the active site or that the inhibitor side chain would have to adopt an alternative binding mode to that observed for other carboxyalkyloxy inhibitors. These data also show that the mDHFR complexes have a decreased active-site volume as reflected in the relative shift of helix C (residues 59–64) by 0.6 Å compared with pcDHFR ternary complexes. These data are consistent with the greater inhibitory potency against pcDHFR.
doi:10.1107/S0907444908022348
PMCID: PMC2615397  PMID: 18703847
dihydrofolate reductase; inhibitors
11.  A general method for phasing novel complex RNA crystal structures without heavy-atom derivatives 
The crystallographic phase problem [Muirhead & Perutz (1963), Nature (London), 199, 633–638] remains the single major impediment to obtaining a three-dimensional structure of a macromolecule once suitable crystals have been obtained. Recently, it was found that it was possible to solve the structure of a 142-nucleotide L1 ligase ribozyme heterodimer that possesses no noncrystallographic symmetry without heavy-atom derivatives, anomalous scattering atoms or other modifications and without a model of the tertiary structure of the ribozyme [Robertson & Scott (2007), Science, 315, 1549–1553]. Using idealized known RNA secondary-structural fragments such as A-form helices and GNRA tetraloops in an iterative molecular-replacement procedure, it was possible to obtain an estimated phase set that, when subjected to solvent flattening, yielded an interpretable electron-density map with minimized model bias, allowing the tertiary structure of the ribozyme to be solved. This approach has also proven successful with other ribozymes, structured RNAs and RNA–protein complexes.
doi:10.1107/S0907444908011578
PMCID: PMC2507861  PMID: 18566509
12.  Alternative models for two crystal structures of bovine rhodopsin 
Two crystal structures of rhodopsin that were originally described using trigonal symmetry can be interpreted in a hexagonal unit cell with a smaller asymmetric unit.
The space-group symmetry of two crystal forms of rhodopsin (PDB codes 1gzm and 2j4y; space group P31) can be re-interpreted as hexagonal (space group P64). Two molecules of the G protein-coupled receptor are present in the asymmetric unit in the trigonal models. However, the noncrystallographic twofold axes parallel to the c axis can be treated as crystallographic symmetry operations in the hexagonal space group. This halves the asymmetric unit and makes all of the protein molecules equivalent in these structures. Corrections for merohedral twinning were also applied in the refinement in the higher symmetry space group for one of the structures (2j4y).
doi:10.1107/S0907444908017162
PMCID: PMC2483493  PMID: 18645239
alternate space groups; rhodopsin; G protein-coupled receptors; integral membrane proteins
13.  Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase 
The short hydrogen bonds in rhamnogalacturonan acetylesterase have been investigated by structure determination of an active-site mutant, 1H NMR spectra and computational methods. Comparisons are made to database statistics. A very short carboxylic acid carboxylate hydrogen bond, buried in the protein, could explain the low-field (18 p.p.m.) 1H NMR signal.
An extremely low-field signal (at approximately 18 p.p.m.) in the 1H NMR spectrum of rhamnogalacturonan acetylesterase (RGAE) shows the presence of a short strong hydrogen bond in the structure. This signal was also present in the mutant RGAE D192N, in which Asp192, which is part of the catalytic triad, has been replaced with Asn. A careful analysis of wild-type RGAE and RGAE D192N was conducted with the purpose of identifying possible candidates for the short hydrogen bond with the 18 p.p.m. deshielded proton. Theor­etical calculations of chemical shift values were used in the interpretation of the experimental 1H NMR spectra. The crystal structure of RGAE D192N was determined to 1.33 Å resolution and refined to an R value of 11.6% for all data. The structure is virtually identical to the high-resolution (1.12 Å) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop. Searches of the Cambridge Structural Database were conducted to obtain information on the donor–acceptor distances of different types of hydrogen bonds. The short hydrogen-bond inter­actions found in RGAE have equivalents in small-molecule structures. An examination of the short hydrogen bonds in RGAE, the calculated pK a values and solvent-accessibilities identified a buried carboxylic acid carboxylate hydrogen bond between Asp75 and Asp87 as the likely origin of the 18 p.p.m. signal. Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB. The shortest hydrogen bonds in RGAE are all located close to the active site and short interactions between Ser and Thr side-chain OH groups and backbone carbonyl O atoms seem to play an important role in the stability of the protein structure. These results illustrate the significance of short strong hydrogen bonds in proteins.
doi:10.1107/S0907444908017083
PMCID: PMC2483496  PMID: 18645234
short hydrogen bonds; low-field NMR signals; rhamnogalacturonan acetylesterase
14.  Representation of viruses in the remediated PDB archive 
A new data model for PDB entries of viruses and other biological assemblies with regular noncrystallographic symmetry is described.
A new scheme has been devised to represent viruses and other biological assemblies with regular noncrystallographic symmetry in the Protein Data Bank (PDB). The scheme describes existing and anticipated PDB entries of this type using generalized descriptions of deposited and experimental coordinate frames, symmetry and frame transformations. A simplified notation has been adopted to express the symmetry generation of assemblies from deposited coordinates and matrix operations describing the required point, helical or crystallographic symmetry. Complete correct information for building full assemblies, subassemblies and crystal asymmetric units of all virus entries is now available in the remediated PDB archive.
doi:10.1107/S0907444908017393
PMCID: PMC2677383  PMID: 18645236
virus structures; Protein Data Bank; database integration; uniform curation; point symmetry; helical symmetry; biological assemblies
15.  Exploiting the anisotropy of anomalous scattering boosts the phasing power of SAD and MAD experiments 
It is shown that the anisotropy of anomalous scattering (AAS) is a significant and ubiquitous effect in data sets collected at an absorption edge and that its exploitation can substantially enhance the phasing power of single- or multi-wavelength anomalous diffraction. The improvements in the phases are typically of the same order of magnitude as those obtained in a conventional approach by adding a second-wavelength data set to a SAD experiment.
The X-ray polarization anisotropy of anomalous scattering in crystals of brominated nucleic acids and selenated proteins is shown to have significant effects on the diffraction data collected at an absorption edge. For conventionally collected single- or multi-wavelength anomalous diffraction data, the main manifestation of the anisotropy of anomalous scattering is the breakage of the equivalence between symmetry-related reflections, inducing intensity differences between them that can be exploited to yield extra phase information in the structure-solution process. A new formalism for describing the anisotropy of anomalous scattering which allows these effects to be incorporated into the general scheme of experimental phasing methods using an extended Harker construction is introduced. This requires a paradigm shift in the data-processing strategy, since the usual separation of the data-merging and phasing steps is abandoned. The data are kept unmerged down to the Harker construction, where the symmetry-breaking is explicitly modelled and refined and becomes a source of supplementary phase information. These ideas have been implemented in the phasing program SHARP. Refinements using actual data show that exploitation of the anisotropy of anomalous scattering can deliver substantial extra phasing power compared with conventional approaches using the same raw data. Examples are given that show improvements in the phases which are typically of the same order of magnitude as those obtained in a conventional approach by adding a second-wavelength data set to a SAD experiment. It is argued that such gains, which come essentially for free, i.e. without the collection of new data, are highly significant, since radiation damage can frequently preclude the collection of a second-wavelength data set. Finally, further developments in synchrotron instrumentation and in the design of data-collection strategies that could help to maximize these gains are outlined.
doi:10.1107/S0907444908010202
PMCID: PMC2467528  PMID: 18566507
anisotropy of anomalous scattering; phasing; SAD; MAD; polarized resonant diffraction
16.  A general method for phasing novel complex RNA crystal structures without heavy-atom derivatives 
Using idealized known RNA secondary-structural fragments, it is demonstrated that it is possible to solve novel complex RNA structures without resort to heavy-atom phasing methods.
The crystallographic phase problem [Muirhead & Perutz (1963 ▶), Nature (London), 199, 633–638] remains the single major impediment to obtaining a three-dimensional structure of a macromolecule once suitable crystals have been obtained. Recently, it was found that it was possible to solve the structure of a 142-nucleotide L1 ligase ribozyme heterodimer that possesses no noncrystallographic symmetry without heavy-atom derivatives, anomalous scattering atoms or other modifications and without a model of the tertiary structure of the ribozyme [Robertson & Scott (2007 ▶), Science, 315, 1549–1553]. Using idealized known RNA secondary-structural fragments such as A-form helices and GNRA tetraloops in an iterative molecular-replacement procedure, it was possible to obtain an estimated phase set that, when subjected to solvent flattening, yielded an interpretable electron-density map with minimized model bias, allowing the tertiary structure of the ribozyme to be solved. This approach has also proven successful with other ribozymes, structured RNAs and RNA–protein complexes.
doi:10.1107/S0907444908011578
PMCID: PMC2507861  PMID: 18566509
RNA; phase problem; molecular replacement
17.  Modulation of activity by Arg407: structure of a fungal α-1,2-mannosidase in complex with a substrate analogue 
Class I α-mannosidases (glycoside hydrolase family GH47) play key roles in the maturation of N-glycans and the ER-associated degradation of unfolded glycoproteins. The 1.95 Å resolution structure of a fungal α-1,2-mannosidase in complex with the substrate analogue methyl-α-D-lyxopyranosyl-(1′,2)-α-D-mannopyranoside (LM) shows the intact disaccharide spanning the −1/+1 subsites, with the D-lyxoside ring in the −1 subsite in the 1C4 chair conformation, and provides insight into the mechanism of catalysis. The absence of the C5′ hydroxymethyl group on the D-lyxoside moiety results in the side chain of Arg407 adopting two alternative conformations: the minor one interacting with Asp375 and the major one interacting with both the D-lyxoside and the catalytic base Glu409, thus disrupting its function. Chemical modification of Asp375 has previously been shown to inactivate the enzyme. Taken together, the data suggest that Arg407, which belongs to the conserved sequence motif RPExxE, may act to modulate the activity of the enzyme. The proposed mechanism for modulating the activity is potentially a general mechanism for this superfamily.
doi:10.1107/S0907444907065572
PMCID: PMC2430470  PMID: 18323617
18.  Iterative-build OMIT maps: map improvement by iterative model building and refinement without model bias 
A procedure for carrying out iterative model building, density modification and refinement is presented in which the density in an OMITregion is essentially unbiased by an atomic model. Density from a set of overlapping OMIT regions can be combined to create a composite ‘iterative-build’ OMIT map that is everywhere unbiased by an atomic model but also everywhere benefiting from the model-based information present elsewhere in the unit cell. The procedure may have applications in the validation of specific features in atomic models as well as in overall model validation. The procedure is demonstrated with a molecular-replacement structure and with an experimentally phased structure and a variation on the method is demonstrated by removing model bias from a structure from the Protein Data Bank.
doi:10.1107/S0907444908004319
PMCID: PMC2424225  PMID: 18453687
19.  Structural characterization of a human Fc fragment engineered for lack of effector functions 
Human Fc fragments containing the L234F/L235E/P331S triple mutation exhibit a dramatic decrease in their binding to several effector molecules (CD64, CD32A, CD16 and C1q). The three-dimensional structure of such a mutated fragment reveals that these broad-ranging functional effects are not caused by major structural rearrangements in the Fc moiety.
The first three-dimensional structure of a human Fc fragment genetically engineered for the elimination of its ability to mediate antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity is reported. When introduced into the lower hinge and CH2 domain of human IgG1 molecules, the triple mutation L234F/L235E/P331S (‘TM’) causes a profound decrease in their binding to human CD64, CD32A, CD16 and C1q. Enzymatically produced Fc/TM fragment was crystallized and its structure was solved at a resolution of 2.3 Å using molecular replacement. This study revealed that the three-dimensional structure of Fc/TM is very similar to those of other human Fc fragments in the experimentally visible region spanning residues 236–­445. Thus, the dramatic broad-ranging effects of TM on IgG binding to several effector molecules cannot be explained in terms of major structural rearrangements in this portion of the Fc.
doi:10.1107/S0907444908007877
PMCID: PMC2467532  PMID: 18560159
Fc fragments
20.  Structure of mouse IP-10, a chemokine 
The structure of mouse IP-10 shows a novel tetrameric association.
Interferon-γ-inducible protein (IP-10) belongs to the CXC class of chemokines and plays a significant role in the patho­physiology of various immune and inflammatory responses. It is also a potent angiostatic factor with antifibrotic properties. The biological activities of IP-10 are exerted by interactions with the G-protein-coupled receptor CXCR3 expressed on Th1 lymphocytes. IP-10 thus forms an attractive target for structure-based rational drug design of anti-inflammatory molecules. The crystal structure of mouse IP-­10 has been determined and reveals a novel tetrameric association. In the tetramer, two conventional CXC chemokine dimers are associated through their N-terminal regions to form a 12-­stranded elongated β-sheet of ∼90 Å in length. This association differs significantly from the previously studied tetramers of human IP-10, platelet factor 4 and neutrophil-activating peptide-2. In addition, heparin- and receptor-binding residues were mapped on the surface of IP-­10 tetramer. Two heparin-binding sites were observed on the surface and were present at the interface of each of the two β-­sheet dimers. The structure supports the formation of higher order oligomers of IP-10, as observed in recent in vivo studies with mouse IP-10, which will have functional relevance.
doi:10.1107/S0907444908007026
PMCID: PMC2665906  PMID: 18560148
interferon-γ-inducible protein; chemokines
21.  Iterative-build OMIT maps: map improvement by iterative model building and refinement without model bias 
An OMIT procedure is presented that has the benefits of iterative model building density modification and refinement yet is essentially unbiased by the atomic model that is built.
A procedure for carrying out iterative model building, density modification and refinement is presented in which the density in an OMIT region is essentially unbiased by an atomic model. Density from a set of overlapping OMIT regions can be combined to create a composite ‘iterative-build’ OMIT map that is everywhere unbiased by an atomic model but also everywhere benefiting from the model-based information present elsewhere in the unit cell. The procedure may have applications in the validation of specific features in atomic models as well as in overall model validation. The procedure is demonstrated with a molecular-replacement structure and with an experimentally phased structure and a variation on the method is demonstrated by removing model bias from a structure from the Protein Data Bank.
doi:10.1107/S0907444908004319
PMCID: PMC2424225  PMID: 18453687
model building; model validation; macromolecular models; Protein Data Bank; refinement; OMIT maps; bias; structure refinement; PHENIX
22.  A vault ribonucleoprotein particle exhibiting 39-fold dihedral symmetry 
A vault from rat liver was crystallized in space group C2. Rotational symmetry searches indicated that the particle has 39-fold dihedral symmetry.
Vault is a 12.9 MDa ribonucleoprotein particle with a barrel-like shape, two protruding caps and an invaginated waist structure that is highly conserved in a wide variety of eukaryotes. Multimerization of the major vault protein (MVP) is sufficient to assemble the entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, vault poly(ADP-ribose) polymerase (VPARP) and telomerase-associated protein 1 (TEP1), as well as a small vault RNA (vRNA), are also associated with vault. Here, the crystallization of vault particles is reported. The crystals belong to space group C2, with unit-cell parameters a = 708.0, b = 385.0, c = 602.9 Å, β = 124.8°. Rotational symmetry searches based on the R factor and correlation coefficient from noncrystallographic symmetry (NCS) averaging indicated that the particle has 39-fold dihedral symmetry.
doi:10.1107/S0907444908004277
PMCID: PMC2467523  PMID: 18453688
vault; ribonucleoproteins
23.  Use of complementary cation and anion heavy-atom salt derivatives to solve the structure of cytochrome P450 46A1 
Crystallization and analysis of the MIRAS heavy-atom structure solution of human cytochrome P450 46A1 using NaI and CsCl quick soaks.
Human cytochrome P450 46A1 (CYP46A1) is one of the key enzymes in cholesterol homeostasis in the brain. The crystallization and heavy-atom structure solution of an active truncated CYP46A1 in complex with the high-affinity substrate analogue cholesterol-3-sulfate (CH-3S) is reported. The 2.6 Å structure of CYP46A1–CH-3S was solved using both anion and cation heavy-atom salts. In addition to the native anomalous signal from the haem iron, an NaI anion halide salt derivative and a complementary CsCl alkali-metal cation salt derivative were used. The general implications of the use of halide and alkali-metal quick soaks are discussed. The importance of using isoionic strength buffers, the titration of heavy-atom salts into different ionic species and the role of concentration are considered. It was observed that cation/anion-binding sites will occasionally overlap, which could negatively impact upon mixed RbBr soaks used for multiple anomalous scatterer MAD (MMAD). The use of complementary cation and anion heavy-atom salt derivatives is a convenient and powerful tool for MIR(AS) structure solution.
doi:10.1107/S0907444908004046
PMCID: PMC2467524  PMID: 18453684
cholesterol sulfate; cholesterol homeostasis; cytochrome P450 46A1; MIRAS; MAD; MIR; heavy-atom derivatives; alkali-metal salts; halide salts
24.  Structure of isochorismate synthase in complex with magnesium 
The structure of the menaquinone-specific isochorismate synthase (MenF) from Escherichia coli has been refined at a resolution of 2.0 Å in complex with magnesium. The magnesium-bound structure has a well defined and organized active site which better represents the active conformation of the enzyme than the currently available structure.
The electron carrier menaquinone is one of many important bacterial metabolites that are derived from the key intermediate chorismic acid. MenF, the first enzyme in the menaquinone pathway, catalyzes the isomerization of chorismate to isochorismate. Here, an improved structure of MenF in a new crystal form is presented. The structure, solved at 2.0 Å resolution in complex with magnesium, reveals a well defined closed active site. Existing evidence suggests that the mechanism of the reaction catalyzed by MenF involves nucleophilic attack of a water molecule on the chorismate ring. The structure reveals a well defined water molecule located in an appropriate position for activation by Lys190 and attack on the substrate.
doi:10.1107/S0907444908005477
PMCID: PMC2467529  PMID: 18453696
chorismate; isochorismate; menaquinone
25.  A knowledge-driven approach for crystallographic protein model completion 
A novel method that uses the conformational distribution of Cα atoms in known structures is used to build short missing regions (‘loops’) in protein models. An initial tree of possible loop paths is pruned according to structural and electron-density criteria and the most likely loop conformation(s) are selected and built.
One of the most cumbersome and time-demanding tasks in completing a protein model is building short missing regions or ‘loops’. A method is presented that uses structural and electron-density information to build the most likely conformations of such loops. Using the distribution of angles and dihedral angles in pentapeptides as the driving parameters, a set of possible conformations for the Cα backbone of loops was generated. The most likely candidate is then selected in a hierarchical manner: new and stronger restraints are added while the loop is built. The weight of the electron-density correlation relative to geometrical considerations is gradually increased until the most likely loop is selected on map correlation alone. To conclude, the loop is refined against the electron density in real space. This is started by using structural information to trace a set of models for the Cα backbone of the loop. Only in later steps of the algorithm is the electron-density correlation used as a criterion to select the loop(s). Thus, this method is more robust in low-density regions than an approach using density as a primary criterion. The algorithm is implemented in a loop-building program, Loopy, which can be used either alone or as part of an automatic building cycle. Loopy can build loops of up to 14 residues in length within a couple of minutes. The average root-mean-square deviation of the Cα atoms in the loops built during validation was less than 0.4 Å. When implemented in the context of automated model building in ARP/wARP, Loopy can increase the completeness of the built models.
doi:10.1107/S0907444908001558
PMCID: PMC2467521  PMID: 18391408
model building; loop modelling; Loopy

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