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1.  Extending the usability of the phasing power of diselenide bonds: SeCys SAD phasing of CsgC using a non-auxotrophic strain 
The CsgC protein is a component of the curli system in Escherichia coli. Reported here is the successful incorporation of selenocysteine (SeCys) and selenomethionine (SeMet) into recombinant CsgC, yielding derivatised crystals suitable for structural determination. Unlike in previous reports, a standard, autotrophic expression strain was used and only single anomalous diffraction (SAD) data were required for successful phasing. The level of SeCys/SeMet incorporation was estimated by mass spectrometry to be about 80%. Native protein crystallised in two different crystal forms (C2221, form 1 and C21, form 2) both diffracting to 2.4 Å, whilst Se-derivatised protein crystallised in C21 and diffracted to 1.7 Å. The Se-derivatised crystals are suitable for SAD structure determination using only anomalous signal derived from the selenocysteine residues. These results extend the usability of SeCys labelling to more general and less favourable cases, rendering it a suitable alternative to traditional phasing approaches.
PMCID: PMC3522112  PMID: 21206057
selenocysteine; SAD; non-auxotrophic E. coli strains; CsgC protein
2.  The 2.6 Å resolution structure of Rhodobacter capsulatus bacterioferritin with metal-free dinuclear site and heme iron in a crystallographic "special position" 
Bacterioferritin from Rhodobacter capsulatus was crystallized and its structure was solved at 2.6 Å resolution. This first structure of a bacterioferritin from a photosynthetic organism is a spherical particle of 24 subunits displaying 432 point group symmetry like ferritin and bacterioferritin from Escherichia coli. Crystallized in the I422 space group, its structural analysis reveals for the first time, the non-symmetric heme molecule located on a two-fold crystallographic symmetry axis. Other hemes of the protomer are situated on two-fold noncrystallographic axes. Apparently both types of sites bind heme in two orientations, leading to an average structure consisting of a symmetric 50:50 mixture, thus satisfying the crystallographic and noncrystallographic symmetry of the crystal. Five water molecules are situated close to the heme which is bound in a hydrophobic pocket and axially coordinated by two crystallographic or noncrystallographically related methionine residues. Its ferroxidase center, in which Fe(II) is oxidized into Fe(III), is empty or fractionally occupied by metal ion. Two positions are observed for the coordinating Glu18 side chain instead of one in the E. coli enzyme in which the site is occupied. This result suggests that the orientation of the Glu18 side chain could be constrained by this interaction.
PMCID: PMC4615704  PMID: 11752777
3.  Phosphates in the Z-DNA dodecamer are flexible, but their P-SAD signal is sufficient for structure solution 
An ultrahigh-resolution structure of the Z-DNA dodecamer, solved from the anomalous signal of P atoms, reveals substantial flexibility of the backbone phosphate groups.
A large number of Z-DNA hexamer duplex structures and a few oligomers of different lengths are available, but here the first crystal structure of the d(CGCGCGCGCGCG)2 dodecameric duplex is presented. Two synchrotron data sets were collected; one was used to solve the structure by the single-wavelength anomalous dispersion (SAD) approach based on the anomalous signal of P atoms, the other set, extending to an ultrahigh resolution of 0.75 Å, served to refine the atomic model to an R factor of 12.2% and an R free of 13.4%. The structure consists of parallel duplexes arranged into practically infinitely long helices packed in a hexagonal fashion, analogous to all other known structures of Z-DNA oligomers. However, the dodecamer molecule shows a high level of flexibility, especially of the backbone phosphate groups, with six out of 11 phosphates modeled in double orientations corresponding to the two previously observed Z-DNA conformations: ZI, with the phosphate groups inclined towards the inside of the helix, and ZII, with the phosphate groups rotated towards the outside of the helix.
PMCID: PMC4089481  PMID: 25004957
Z-DNA structure; Z-DNA dodecamer; phosphorus SAD phasing; ultrahigh resolution; flexibility of phosphate groups
4.  Exploiting subtle structural differences in heavy-atom derivatives for experimental phasing 
Small differences between heavy-atom derivatives can be exploited to improve the experimental phasing by treating each derivative as an independent SAD data set and combining these data sets with circular permutable pairs of SIR data sets. The basis for this generally applicable approach is that the effective resolution of isomorphous signals between highly isomorphous derivatives is often much higher than the effective resolution of the anomalous signals of individual derivative data sets.
Structure determination using the single isomorphous replacement (SIR) or single-wavelength anomalous diffraction (SAD) methods with weak derivatives remains very challenging. In a recent structure determination of glycoprotein E2 from bovine viral diarrhea virus, three isomorphous uranium-derivative data sets were merged to obtain partially interpretable initial experimental maps. Small differences between them were then exploited by treating them as three independent SAD data sets plus three circular pairwise SIR data sets to improve the experimental maps. Here, how such subtle structural differences were exploited for experimental phasing is described in detail. The basis for why this approach works is also provided: the effective resolution of isomorphous signals between highly isomorphous derivatives is often much higher than the effective resolution of the anomalous signals of individual derivative data sets. Hence, the new phasing approaches outlined here will be generally applicable to structure determinations involving weak derivatives.
PMCID: PMC4089484  PMID: 25004964
phasing; heavy-atom derivatives; isomorphous replacement; single anomalous dispersion (SAD); phase combination; data set merging
5.  Structural insights into interactions of C/EBP transcriptional activators with the Taz2 domain of p300 
Crystal contacts in the structure of a chimeric protein composed of residues 1723–1818 of p300 Taz2 and residues 37–61 of C/EBP∊ reveal a possible mode of interactions of C/EBP transcriptional activators with p300/CBP.
Members of the C/EBP family of transcription factors bind to the Taz2 domain of p300/CBP and mediate its phosphorylation through the recruitment of specific kinases. Short sequence motifs termed homology boxes A and B, which comprise their minimal transactivation domains (TADs), are conserved between C/EBP activators and are necessary for specific p300/CBP binding. A possible mode of interaction between C/EBP TADs and the p300 Taz2 domain was implied by the crystal structure of a chimeric protein composed of residues 1723–1818 of p300 Taz2 and residues 37–61 of C/EBP∊. The segment corresponding to the C/EBP∊ TAD forms two orthogonally disposed helices connected by a short linker and interacts with the core structure of Taz2 from a symmetry-related molecule. It is proposed that other members of the C/EBP family interact with the Taz2 domain in the same manner. The position of the C/EBP∊ peptide on the Taz2 protein interaction surface suggests that the N-termini of C/EBP proteins are unbound in the C/EBP–p300 Taz2 complex. This observation is in agreement with the known location of the docking site of protein kinase HIPK2 in the C/EBPβ N-terminus, which associates with the C/EBPβ–p300 complex.
PMCID: PMC4089485  PMID: 25004968
transcription; molecular recognition; ligand binding; HIPK2
6.  Two high-mobility group box domains act together to underwind and kink DNA 
The crystal structure of HMGB1 box A bound to an unmodified AT-rich DNA fragment is reported at a resolution of 2 Å. A new mode of DNA recognition for HMG box proteins is found in which two box A domains bind in an unusual configuration generating a highly kinked DNA structure.
High-mobility group protein 1 (HMGB1) is an essential and ubiquitous DNA architectural factor that influences a myriad of cellular processes. HMGB1 contains two DNA-binding domains, box A and box B, which have little sequence specificity but have remarkable abilities to underwind and bend DNA. Although HMGB1 box A is thought to be responsible for the majority of HMGB1–DNA interactions with pre-bent or kinked DNA, little is known about how it recognizes unmodified DNA. Here, the crystal structure of HMGB1 box A bound to an AT-rich DNA fragment is reported at a resolution of 2 Å. Two box A domains of HMGB1 collaborate in an unusual configuration in which the Phe37 residues of both domains stack together and intercalate the same CG base pair, generating highly kinked DNA. This represents a novel mode of DNA recognition for HMGB proteins and reveals a mechanism by which structure-specific HMG boxes kink linear DNA.
PMCID: PMC4498601  PMID: 26143914
high-mobility group protein; X-ray crystallography; DNA binding; HMGB1
7.  Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment 
The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition.
Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macro­globulin, this protease-activation mechanism is likely to operate across the diverse members of this group.
PMCID: PMC4498604  PMID: 26143919
α-2-macroglobulin; protease inhibitor; conformational change; intrinsic disorder
8.  Structural and biochemical analyses of a Clostridium perfringens sortase D transpeptidase 
The structure of C. perfringens sortase D was determined at 1.99 Å resolution. Comparative biochemical and structural analyses revealed that this transpeptidase may represent a new subclass of the sortase D family.
The assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria is mediated by the sortase family of enzymes. These cysteine transpeptidases catalyze a unique sorting signal motif located at the C-terminus of their target substrate and promote the covalent attachment of these proteins onto an amino nucleophile located on another protein or on the bacterial cell wall. Each of the six distinct classes of sortases displays a unique biological role, with sequential activation of multiple sortases often observed in many Gram-positive bacteria to decorate their peptidoglycans. Less is known about the members of the class D family of sortases (SrtD), but they have a suggested role in spore formation in an oxygen-limiting environment. Here, the crystal structure of the SrtD enzyme from Clostridium perfringens was determined at 1.99 Å resolution. Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded β-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro, with a significant preference for the LPQTGS sorting motif. Additionally, the catalytic activity of SrtD is most efficient at 316 K and can be further improved in the presence of magnesium cations. Since C. perfringens spores are heat-resistant and lead to foodborne illnesses, characterization of the spore-promoting sortase SrtD may lead to the development of new antimicrobial agents.
PMCID: PMC4498605  PMID: 26143922
sortase D; spore-forming Gram-positive bacterium; LPQTGS sorting motif
9.  From bacterial to human dihydrouridine synthase: automated structure determination 
The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template.
The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr_rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.
PMCID: PMC4498606  PMID: 26143927
dihydrouridine synthase; X-ray crystallography; MR-Rosetta; tRNA modification; lung cancer
10.  High-resolution crystal structures of the solubilized domain of porcine cytochrome b 5  
Crystal structures of the solubilized domain of cytochrome b 5 from porcine liver were determined at sub-angstrom resolution in two crystal forms for both the oxidized and reduced states. The high-resolution structures provided information about the factors that are important for regulating the electronic properties of the haem group of cytochrome b 5.
Mammalian microsomal cytochrome b 5 has multiple electron-transfer partners that function in various electron-transfer reactions. Four crystal structures of the solubilized haem-binding domain of cytochrome b 5 from porcine liver were determined at sub-angstrom resolution (0.76–0.95 Å) in two crystal forms for both the oxidized and reduced states. The high-resolution structures clearly displayed the electron density of H atoms in some amino-acid residues. Unrestrained refinement of bond lengths revealed that the protonation states of the haem propionate group may be involved in regulation of the haem redox properties. The haem Fe coordination geometry did not show significant differences between the oxidized and reduced structures. However, structural differences between the oxidized and reduced states were observed in the hydrogen-bond network around the axial ligand His68. The hydrogen-bond network could be involved in regulating the redox states of the haem group.
PMCID: PMC4498607  PMID: 26143928
electron transfer; haem; sub-angstrom resolution
11.  Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6′)-Ie-APH(2′′)-Ia 
The crystal structure of the C-terminal phosphotransferase domain of the bifunctional aminoglycoside acetyltransferase–phosphotransferase AAC(6′)-Ie-APH(2′′)-Ia has been determined to 2.3 Å resolution. The enzyme was crystallized as the Mg2GDP binary complex.
The bifunctional acetyltransferase(6′)-Ie-phosphotransfer­ase(2′′)-Ia [AAC(6′)-Ie-APH(2′′)-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6′)-Ie-APH(2′′)-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2′′)-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2′′)-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2′′)-IIa and APH(2′′)-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2′′)-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2′′)-IIIa enzyme. In APH(2′′)-Ia this GTP selectivity is governed by the presence of a ‘gatekeeper’ residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2′′)-Ia into a dual-specificity enzyme.
PMCID: PMC4051501  PMID: 24914967
AAC(6′)-Ie-APH(2′′)-Ia; aminoglycoside-resistance enzymes
12.  A point mutation in the [2Fe–2S] cluster binding region of the NAF-1 protein (H114C) dramatically hinders the cluster donor properties 
NAF-1 has been shown to be related with human health and disease, is upregulated in epithelial breast cancer and suppression of its expression significantly suppresses tumor growth. It is shown that replacement of the single His ligand with Cys resulted in dramatic changes to the properties of its 2Fe-2S clusters without any global crystal structural changes.
NAF-1 is an important [2Fe–2S] NEET protein associated with human health and disease. A mis-splicing mutation in NAF-1 results in Wolfram Syndrome type 2, a lethal childhood disease. Upregulation of NAF-1 is found in epithelial breast cancer cells, and suppression of NAF-1 expression by knockdown significantly suppresses tumor growth. Key to NAF-1 function is the NEET fold with its [2Fe–2S] cluster. In this work, the high-resolution structure of native NAF-1 was determined to 1.65 Å resolution (R factor = 13.5%) together with that of a mutant in which the single His ligand of its [2Fe–2S] cluster, His114, was replaced by Cys. The NAF-1 H114C mutant structure was determined to 1.58 Å resolution (R factor = 16.0%). All structural differences were localized to the cluster binding site. Compared with native NAF-1, the [2Fe–2S] clusters of the H114C mutant were found to (i) be 25-fold more stable, (ii) have a redox potential that is 300 mV more negative and (iii) have their cluster donation/transfer function abolished. Because no global structural differences were found between the mutant and the native (wild-type) NAF-1 proteins, yet significant functional differences exist between them, the NAF-1 H114C mutant is an excellent tool to decipher the underlying biological importance of the [2Fe–2S] cluster of NAF-1 in vivo.
PMCID: PMC4051502  PMID: 24914968
autophagy; redox potential; 2Fe–2S cluster transfer and cluster stability; breast cancer; WFS2; longevity
13.  Structural and bioinformatic characterization of an Acinetobacter baumannii type II carrier protein 
The high-resolution crystal structure of a free-standing carrier protein from Acinetobacter baumannii that belongs to a larger NRPS-containing operon, encoded by the ABBFA_003406–ABBFA_003399 genes of A. baumannii strain AB307-0294, that has been implicated in A. baumannii motility, quorum sensing and biofilm formation, is presented.
Microorganisms produce a variety of natural products via secondary metabolic biosynthetic pathways. Two of these types of synthetic systems, the nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), use large modular enzymes containing multiple catalytic domains in a single protein. These multidomain enzymes use an integrated carrier protein domain to transport the growing, covalently bound natural product to the neighboring catalytic domains for each step in the synthesis. Interestingly, some PKS and NRPS clusters contain free-standing domains that interact intermolecularly with other proteins. Being expressed outside the architecture of a multi-domain protein, these so-called type II proteins present challenges to understand the precise role they play. Additional structures of individual and multi-domain components of the NRPS enzymes will therefore provide a better understanding of the features that govern the domain interactions in these interesting enzyme systems. The high-resolution crystal structure of a free-standing carrier protein from Acinetobacter baumannii that belongs to a larger NRPS-containing operon, encoded by the ABBFA_003406–ABBFA_003399 genes of A. baumannii strain AB307-0294, that has been implicated in A. baumannii motility, quorum sensing and biofilm formation, is presented here. Comparison with the closest structural homologs of other carrier proteins identifies the requirements for a conserved glycine residue and additional important sequence and structural requirements within the regions that interact with partner proteins.
PMCID: PMC4051507  PMID: 24914982
natural product biosynthesis; peptidyl carrier proteins; acyl carrier proteins; Acinetobacter baumannii; motility; biofilm formation; nonribosomal peptide synthetase
14.  Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II 
The structure of human carbonic anhydrase II in complex with cholate has been determined to 1.54 Å resolution. Elucidation of the novel inhibition mechanism of cholate will aid in the development of a nonsulfur-containing, isoform-specific therapeutic agent.
The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO2 into bicarbonate and a proton. Human isoform CA II (HCA II) is abundant in the surface epithelial cells of the gastric mucosa, where it serves an important role in cytoprotection through bicarbonate secretion. Physiological inhibition of HCA II via the bile acids contributes to mucosal injury in ulcerogenic conditions. This study details the weak biophysical interactions associated with the binding of a primary bile acid, cholate, to HCA II. The X-ray crystallographic structure determined to 1.54 Å resolution revealed that cholate does not make any direct hydrogen-bond interactions with HCA II, but instead reconfigures the well ordered water network within the active site to promote indirect binding to the enzyme. Structural knowledge of the binding interactions of this nonsulfur-containing inhibitor with HCA II could provide the template design for high-affinity, isoform-specific therapeutic agents for a variety of diseases/pathological states, including cancer, glaucoma, epilepsy and osteoporosis.
PMCID: PMC4051509  PMID: 24914985
carbonic anhydrase II; cholate; inhibition
15.  Structure of the LCMV nucleoprotein provides a template for understanding arenavirus replication and immunosuppression 
The crystal structure of the Lymphocytic choriomeningitis virus (LCMV) nucleoprotein C-terminal domain adopts the fold of an exonuclease and provides an important structural template for the study of this prototypic arenavirus.
The X-ray crystal structure of the Lymphocytic chorio­meningitis virus nucleoprotein C-terminal immunosuppressive domain (LCMV NPΔ340) was determined to 2.0 Å resolution. The structure indicates that LCMV NPΔ340, like the other structurally characterized arenaviral nucleoproteins, adopts the fold of an exonuclease. This structure provides a crucial three-dimensional template for functional exploration of the replication and immunosuppression of this prototypic arenavirus.
PMCID: PMC4051510  PMID: 24914986
Lymphocytic choriomeningitis virus; nucleoprotein
16.  3-Sulfinopropionyl-coenzyme A (3SP-CoA) desulfinase from Advenella mimigardefordensis DPN7T: crystal structure and function of a desulfinase with an acyl-CoA dehydrogenase fold 
3-Sulfinopropionyl-coenzyme A desulfinase (AcdDPN7) is a novel desulfinase which catalyzes sulfur abstraction in the catabolic pathway of 3,3′-dithiodipropionic acid. The crystal structures of native AcdDPN7 at 1.89 Å resolution and of native AcdDPN7 soaked with the substrate analogue succinyl-CoA at 2.30 Å resolution revealed an acyl-CoA dehydrogenase fold with Arg84 as a key residue in the desulfination reaction.
3-Sulfinopropionyl-coenzyme A (3SP-CoA) desulfinase (AcdDPN7; EC was identified during investigation of the 3,3′-dithiodipropionic acid (DTDP) catabolic pathway in the betaproteobacterium Advenella mimigardefordensis strain DPN7T. DTDP is an organic disulfide and a precursor for the synthesis of polythioesters (PTEs) in bacteria, and is of interest for biotechnological PTE production. AcdDPN7 catalyzes sulfur abstraction from 3SP-CoA, a key step during the catabolism of DTDP. Here, the crystal structures of apo AcdDPN7 at 1.89 Å resolution and of its complex with the CoA moiety from the substrate analogue succinyl-CoA at 2.30 Å resolution are presented. The apo structure shows that AcdDPN7 belongs to the acyl-CoA dehydrogenase superfamily fold and that it is a tetramer, with each subunit containing one flavin adenine dinucleotide (FAD) molecule. The enzyme does not show any dehydrogenase activity. Dehydrogenase activity would require a catalytic base (Glu or Asp residue) at either position 246 or position 366, where a glutamine and a glycine are instead found, respectively, in this desulfinase. The positioning of CoA in the crystal complex enabled the modelling of a substrate complex containing 3SP-CoA. This indicates that Arg84 is a key residue in the desulfination reaction. An Arg84Lys mutant showed a complete loss of enzymatic activity, suggesting that the guanidinium group of the arginine is essential for desulfination. AcdDPN7 is the first desulfinase with an acyl-CoA dehydrogenase fold to be reported, which underlines the versatility of this enzyme scaffold.
PMCID: PMC4461206  PMID: 26057676
desulfinase; dehydrogenase fold; 3-sulfinopropionyl-coenzyme A; 3,3′-dithiodipropionic acid; arginine
17.  A revised partiality model and post-refinement algorithm for X-ray free-electron laser data 
An updated partiality model and post-refinement algorithm for XFEL snapshot diffraction data is presented and confirmed by observing anomalous density for S atoms at an X-ray wavelength of 1.3 Å.
Research towards using X-ray free-electron laser (XFEL) data to solve structures using experimental phasing methods such as sulfur single-wavelength anomalous dispersion (SAD) has been hampered by shortcomings in the diffraction models for X-ray diffraction from FELs. Owing to errors in the orientation matrix and overly simple partiality models, researchers have required large numbers of images to converge to reliable estimates for the structure-factor amplitudes, which may not be feasible for all biological systems. Here, data for cytoplasmic polyhedrosis virus type 17 (CPV17) collected at 1.3 Å wavelength at the Linac Coherent Light Source (LCLS) are revisited. A previously published definition of a partiality model for reflections illuminated by self-amplified spontaneous emission (SASE) pulses is built upon, which defines a fraction between 0 and 1 based on the intersection of a reflection with a spread of Ewald spheres modelled by a super-Gaussian wavelength distribution in the X-ray beam. A method of post-refinement to refine the parameters of this model is suggested. This has generated a merged data set with an overall discrepancy (by calculating the R split value) of 3.15% to 1.46 Å resolution from a 7225-image data set. The atomic numbers of C, N and O atoms in the structure are distinguishable in the electron-density map. There are 13 S atoms within the 237 residues of CPV17, excluding the initial disordered methionine. These only possess 0.42 anomalous scattering electrons each at 1.3 Å wavelength, but the 12 that have single predominant positions are easily detectable in the anomalous difference Fourier map. It is hoped that these improvements will lead towards XFEL experimental phase determination and structure determination by sulfur SAD and will generally increase the utility of the method for difficult cases.
PMCID: PMC4461207  PMID: 26057680
post-refinement; free-electron laser; partiality
18.  Structure determination of an integral membrane protein at room temperature from crystals in situ  
The X-ray structure determination of an integral membrane protein using synchrotron diffraction data measured in situ at room temperature is demonstrated.
The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.
PMCID: PMC4461203  PMID: 26057664
in situ data collection; membrane protein; multiple data sets; synchrotron beamline
19.  In meso in situ serial X-ray crystallography of soluble and membrane proteins 
A method for performing high-throughput in situ serial X-ray crystallography with soluble and membrane proteins in the lipid cubic phase is described. It works with microgram quantities of protein and lipid (and ligand when present) and is compatible with the most demanding sulfur SAD phasing.
The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the β2-adrenoreceptor–Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development.
PMCID: PMC4461204  PMID: 26057665
AlgE; bromine SAD; experimental phasing; in meso; in situ; lipid cubic phase; membrane protein; mesophase; PepTSt; sulfur SAD; serial crystallography
20.  Structural bases for N-glycan processing by mannoside phosphorylase 
Crystal structures of the GH130 enzyme Uhgb_MP in the apo form and in complex with mannose and N-acetylglucosamine are described and the structural determinants of the functional specificities of the enzymes involved in N-glycan breakdown by human gut bacteria are identified.
The first crystal structure of Uhgb_MP, a β-1,4-mannopyranosyl-chitobiose phosphorylase belonging to the GH130 family which is involved in N-glycan degradation by human gut bacteria, was solved at 1.85 Å resolution in the apo form and in complex with mannose and N-acetylglucosamine. SAXS and crystal structure analysis revealed a hexameric structure, a specific feature of GH130 enzymes among other glycoside phosphorylases. Mapping of the −1 and +1 subsites in the presence of phosphate confirmed the conserved Asp104 as the general acid/base catalytic residue, which is in agreement with a single-step reaction mechanism involving Man O3 assistance for proton transfer. Analysis of this structure, the first to be solved for a member of the GH130_2 subfamily, revealed Met67, Phe203 and the Gly121–Pro125 loop as the main determinants of the specificity of Uhgb_MP and its homologues towards the N-glycan core oligosaccharides and mannan, and the molecular bases of the key role played by GH130 enzymes in the catabolism of dietary fibre and host glycans.
PMCID: PMC4461205  PMID: 26057673
GH130 enzymes; N-glycans; glycoside phosphorylases; human gut microbiota
21.  Accurate macromolecular crystallographic refinement: incorporation of the linear scaling, semiempirical quantum-mechanics program DivCon into the PHENIX refinement package 
Semiempirical quantum-chemical X-ray macromolecular refinement using the program DivCon integrated with PHENIX is described.
Macromolecular crystallographic refinement relies on sometimes dubious stereochemical restraints and rudimentary energy functionals to ensure the correct geometry of the model of the macromolecule and any covalently bound ligand(s). The ligand stereochemical restraint file (CIF) requires a priori understanding of the ligand geometry within the active site, and creation of the CIF is often an error-prone process owing to the great variety of potential ligand chemistry and structure. Stereochemical restraints have been replaced with more robust functionals through the integration of the linear-scaling, semiempirical quantum-mechanics (SE-QM) program DivCon with the PHENIX X-ray refinement engine. The PHENIX/DivCon package has been thoroughly validated on a population of 50 protein–ligand Protein Data Bank (PDB) structures with a range of resolutions and chemistry. The PDB structures used for the validation were originally refined utilizing various refinement packages and were published within the past five years. PHENIX/DivCon does not utilize CIF(s), link restraints and other parameters for refinement and hence it does not make as many a priori assumptions about the model. Across the entire population, the method results in reasonable ligand geometries and low ligand strains, even when the original refinement exhibited difficulties, indicating that PHENIX/DivCon is applicable to both single-structure and high-throughput crystallography.
PMCID: PMC4014119  PMID: 24816093
X-ray crystallography; quantum-mechanics refinement; regional refinement; stereochemical restraints; ligand strain; high-throughput crystallography
22.  Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility 
This work reports crystal structures of trimeric riboflavin synthase from the pathogen B. abortus both as the apo protein and in complex with several ligands of interest. It is shown that ligand binding drives the assembly of the unique active site of the trimer, and these findings are complemented by a detailed kinetic study on this enzyme, in which marked inhibition by substrate and product was observed.
Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C 3 symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.
PMCID: PMC4014124  PMID: 24816110
enzyme–ligand complex; inhibition by substrate and product; 6,7-dimethyl-8-ribityllumazine; vitamin B2
23.  Diamonds in the rough: a strong case for the inclusion of weak-intensity X-ray diffraction data 
Here, new evidence is provided to show that the inclusion of weak-intensity, high-resolution X-ray diffraction data helps to improve the quality of experimental phases by imposing proper constraints on electron-density models during noncrystallographic symmetry averaging.
Overwhelming evidence exists to show that the inclusion of weak-intensity, high-resolution X-ray diffraction data helps improve the refinement of atomic models by imposing strong constraints on individual and overall temperature B factors and thus the quality of crystal structures. Some researchers consider these data to be of little value and opt to discard them during data processing, particularly at medium and low resolution, at which individual B factors of atomic models cannot be refined. Here, new evidence is provided to show that the inclusion of these data helps to improve the quality of experimental phases by imposing proper constraints on electron-density models during noncrystallographic symmetry (NCS) averaging. Using electron-density correlation coefficients as criteria, the resolution of data has successfully been extended from 3.1 to 2.5 Å resolution with redundancy-independent merging R factors from below 100% to about 310%. It is further demonstrated that phase information can be fully extracted from observed amplitudes through de novo NCS averaging. Averaging starts with uniform density inside double-shelled spherical masks and NCS matrices that are derived from bound heavy-atom clusters at the vertices of cuboctahedrally symmetric protein particles.
PMCID: PMC4014128  PMID: 24816117
weak-intensity data; refinement; YfbU
24.  Using support vector machines to improve elemental ion identification in macromolecular crystal structures 
A method to automatically identify possible elemental ions in X-ray crystal structures has been extended to use support vector machine (SVM) classifiers trained on selected structures in the PDB, with significantly improved sensitivity over manually encoded heuristics.
In the process of macromolecular model building, crystallographers must examine electron density for isolated atoms and differentiate sites containing structured solvent molecules from those containing elemental ions. This task requires specific knowledge of metal-binding chemistry and scattering properties and is prone to error. A method has previously been described to identify ions based on manually chosen criteria for a number of elements. Here, the use of support vector machines (SVMs) to automatically classify isolated atoms as either solvent or one of various ions is described. Two data sets of protein crystal structures, one containing manually curated structures deposited with anomalous diffraction data and another with automatically filtered, high-resolution structures, were constructed. On the manually curated data set, an SVM classifier was able to distinguish calcium from manganese, zinc, iron and nickel, as well as all five of these ions from water molecules, with a high degree of accuracy. Additionally, SVMs trained on the automatically curated set of high-resolution structures were able to successfully classify most common elemental ions in an independent validation test set. This method is readily extensible to other elemental ions and can also be used in conjunction with previous methods based on a priori expectations of the chemical environment and X-ray scattering.
PMCID: PMC4427199  PMID: 25945580
elemental ion identification; support vector machines; model building
25.  Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus  
The purified putative betaine aldehyde dehydrogenase SACOL2628 from the early methicillin-resistant S. aureus COL has betaine aldehyde dehydrogenase activity and is structurally similar to aldehyde dehydrogenases.
When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD+) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD+, NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.
PMCID: PMC4427200  PMID: 25945581
betaine aldehyde dehydrogenase; Staphylococcus aureus; structural genomics; high-throughput approach; infectious diseases

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