Polyethylene glycol (PEG) is often used in protein crystallography as a low ionic strength precipitant for crystallization and a cryoprotectant for low temperature data collection. Prompted by the discovery of an apparent L-lactate molecule bound in the active site of the E. coli PutA proline dehydrogenase domain crystal structure, we measured the L-lactate concentration of several PEG solutions. Fifty percent (w/v) solutions of PEGs with molecular weight 3000, 4000, and 8000 contain millimolar levels of L-lactate. In contrast, L-lactate was not detected in solutions of PEG monomethyl ethers or PEG 3350. These results help explain why L-lactate was present in the proline dehydrogenase domain crystal structure. This work also has implications for the crystallization of enzymes that bind L-lactate.
Rubidium is a monovalent metal that can be used as a counterion in protein solutions. X-ray anomalous scattering from rubidium ions bound to the protein surface was used for phasing of the crystal structure of the hsp60 apical domain from Thermus thermophilus. Multiple-wavelength anomalous dispersion (MAD) data were collected from a crystal obtained from a solution containing 0.2 M rubidium salt. One molecule of protein (147 amino acids) binds one well ordered and one poorly ordered Rb atom. Phases calculated with the program SHARP were sufficient for automatic tracing and side-chain assignment using the program ARP/wARP. The data show that bound rubidium ions can be used to determine protein structures and to study the interaction of monovalent metal ions with proteins and other macromolecules.
Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species. These protozoa cause serious diseases for which current therapies are inadequate. High-resolution structures have been determined, using data between 1.6 and 1.1 Å resolution, of T. brucei PTR1 in complex with pemetrexed, trimetrexate, cyromazine and a 2,4-diaminopyrimidine derivative. The structures provide insight into the interactions formed by new molecular entities in the enzyme active site with ligands that represent lead compounds for structure-based inhibitor development and to support early-stage drug discovery.
The GTPases Ffh and FtsY are components of the prokaryotic signal recognition particle protein-targeting pathway. The two proteins interact in a GTP-dependent manner, forming a complex that can be stabilized by use of the non-hydrolyzable GTP analog GMPPCP. Crystals of the complex of the NG GTPase domains of the two proteins have been obtained from ammonium sulfate solutions. Crystals grow with several different morphologies, predominately as poorly diffracting plates and needle clusters, but occasionally as well diffracting rods. It has been demonstrated that all forms of the crystals observed contain an intact complex. Diffraction data to 2.0 Å resolution have been measured.
Two new structures of the SRP GTPase Ffh have been determined at 1.1 Å resolution and provide the basis for comparative examination of the extensive water structure of the apo conformation of these GTPases. A set of well defined water-binding positions have been identified in the active site of the two-domain ‘NG’ GTPase, as well as at two functionally important interfaces. The water hydrogen-bonding network accommodates alternate conformations of the protein side chains by undergoing local rearrangements and, in one case, illustrates binding of a solute molecule within the active site by displacement of water molecules without further disruption of the water-interaction network. A subset of the water positions are well defined in several lower resolution structures, including those of different nucleotide-binding states; these appear to function in maintaining the protein structure. Consistent arrangements of surface water between three different ultrahigh-resolution structures provide a framework for beginning to understand how local water structure contributes to protein–ligand and protein–protein binding in the SRP GTPases.
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.
selenocysteine; SAD; non-auxotrophic E. coli strains; CsgC protein
During X-ray irradiation protein crystals radiate energy in the form of small amounts of visible light. This is known as X-ray-excited optical luminescence (XEOL). The XEOL of several proteins and their constituent amino acids has been characterized using the microspectrophotometers at the Swiss Light Source and Diamond Light Source. XEOL arises primarily from aromatic amino acids, but the effects of local environment and quenching within a crystal mean that the XEOL spectrum of a crystal is not the simple sum of the spectra of its constituent parts. Upon repeated exposure to X-rays XEOL spectra decay non-uniformly, suggesting that XEOL is sensitive to site-specific radiation damage. However, rates of XEOL decay were found not to correlate to decays in diffracting power, making XEOL of limited use as a metric for radiation damage to protein crystals.
The problems encountered during the phasing and structure determination of the packaging enzyme P4 from bacteriophage ϕ13 using the anomalous signal from selenium in a single-wavelength anomalous dispersion experiment (SAD) are described. The oligomeric state of P4 in the virus is a hexamer (with sixfold rotational symmetry) and it crystallizes in space group C2, with four hexamers in the crystallographic asymmetric unit. Current state-of-the-art ab initio phasing software yielded solutions consisting of 96 atoms arranged as sixfold symmetric clusters of Se atoms. However, although these solutions showed high correlation coefficients indicative that the substructure had been solved, the resulting phases produced uninterpretable electron-density maps. Only after further analysis were correct solutions found (also of 96 atoms), leading to the eventual identification of the positions of 120 Se atoms. Here, it is demonstrated how the difficulties in finding a correct phase solution arise from an intricate false-minima problem.
Angiogenin is an unusual member of the pancreatic ribonuclease
superfamily that induces blood vessel formation and is a promising anticancer
target. The three-dimensional structure of murine angiogenin (mAng) has been
determined by X-ray crystallography. Two structures are presented, one a complex
with sulphate ions (1.5 Å-resolution) and the other with phosphate
ions (1.6 Å-resolution). Residues forming the putative B1,
P1 and B2 subsites occupy positions similar to their
hAng counterparts and are likely to play similar roles. The anions occupy the
P1 subsite, sulphate binding conventionally and phosphate
adopting two orientations, one of which is novel. The B1 subsite is
obstructed by Glu116 and Phe119, with the latter assuming a less invasive
position than its hAng counterpart. Hydrophobic interactions between the
C-terminal segment and the main body of the protein are more extensive than in
hAng and may underly the lower enzymatic activity of the murine protein.
Elsewhere, the structure of the H3–B2 loop supports the view that
hAng Asn61 interacts directly with cell surface molecules and does not merely
stabilize adjacent regions of the hAng structure. mAng crystals appear to offer
small-molecule inhibitors a clear route to the active site and may even
withstand a reorientation of the C-terminal segment that provides access to the
cryptic B1 subsite. These features represent considerable advantages
over crystalline hAng and bAng.
A new cellular growth factor termed augmenter of liver regeneration (ALR) has been crystallized, ALR has been shown to have a proliferative effect on liver cells while at the same time producing an immunosuppressive effect on liver-resident natural killer cells and liver-resident mononuclear leukocytes. In addition, ALR appears to play an important role in the synthesis and stabilization of mitochondrial gene transcripts in, actively regenerating cells. ALR crystals diffract to beyond 2Å resolution and belong to space group P21212, with a = 125.1, b = 108.1 and c = 38.5Å. Based on four molecules per asymmetric unit, the Matthews coefficient is calculated to be 2.16Å3Da−1 which corresponds to a solvent content of 43 %.
This paper presents a methodology to obtain candidate conformations of multidomain proteins for use in Molecular Replacement. For each separate domain, orientational relationship between the template and the target structure is obtained by using MR. Then, the orientational relationships of the domains are used to calculate the relative rotation between those domains in the target conformation by using pose estimation techniques from the field of Robotics and Computer Vision. With the angle of relative rotation between the domains as a cost function, iterative normal mode analysis is used to drive the template structure into the candidate conformation to match X-ray crystallography data obtained for the target conformation. As a validation, the proposed method is applied to three test proteins: Ribose-binding protein; Lactoferrin; and Calcium ATPase. In each test case, the orientation and translation of the final candidate conformation are generated correctly from the suggested procedure. The results show that the proposed method can yield applicable candidate conformations for MR and reveal the structural details of the target conformation and its position and orientation in the crystallographic unit cell.
High-throughput (HT) protein crystallography is severely impeded by the relatively low success rate of protein crystallization. Proteins whose structures are not solved in the HT pipeline owing to attrition in any phase of the project are referred to as the high-hanging fruit, in contrast to those proteins that yielded good-quality crystals and crystal structures, which are referred to as low-hanging fruit. It has previously been shown that proteins that do not crystallize in the wild-type form can have their surfaces engineered by site-directed mutagenesis in order to create patches of low conformational entropy that are conducive to forming intermolecular interactions. The application of this method to selected proteins from the Bacillus subtilis genome which failed to crystallize in the HT mode is now reported. In this paper, the crystal structure of the product of the YdeN gene is reported. Of three prepared double mutants, i.e. E124A/K127A, E167A/E169A and K88A/Q89A, the latter gave high-quality crystals and the crystal structure was solved by SAD at 1.8 Å resolution. The protein is a canonical α/β hydrolase, with an active site that is accessible to solvent.
The automation of protein structure determination is an essential component for high-throughput structural analysis in protein X-ray crystallography and is a key element in structural genomics. This highly challenging undertaking relies at present on the availability of high-quality native and derivatized protein crystals diffracting to high or moderate resolution, respectively. Obtaining such crystals often requires significant effort. The present study demonstrates that phases obtained at low resolution (>3.0 Å) from crystals of SeMet-labeled protein can be successfully used for automated structure determination. The crystal structure of acetate CoA-transferase α-subunit was solved using 3.4 Å multiwavelength anomalous dispersion data collected from a crystal containing SeMet-substituted protein and 1.9 Å data collected from a native protein crystal.
The structure of the YlxR protein of unknown function from Streptococcus pneumonia was determined to 1.35 Å. YlxR is expressed from the nusA/infB operon in bacteria and belongs to a small protein family (COG2740) that shares a conserved sequence motif GRGA(Y/W). The family shows no significant amino-acid sequence similarity with other proteins. Three-wavelength diffraction MAD data were collected to 1.7 Å from orthorhombic crystals using synchrotron radiation and the structure was determined using a semi-automated approach. The YlxR structure resembles a two-layer α/β sandwich with the overall shape of a cylinder and shows no structural homology to proteins of known structure. Structural analysis revealed that the YlxR structure represents a new protein fold that belongs to the α–β plait superfamily. The distribution of the electrostatic surface potential shows a large positively charged patch on one side of the protein, a feature often found in nucleic acid-binding proteins. Three sulfate ions bind to this positively charged surface. Analysis of potential binding sites uncovered several substantial clefts, with the largest spanning 3/4 of the protein. A similar distribution of binding sites and a large sharply bent cleft are observed in RNA-binding proteins that are unrelated in sequence and structure. It is proposed that YlxR is an RNA-binding protein.
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.
antibody; immunoglobulin; unglycosylated CH2, IgG; Fc; CH2 domain
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.
Photoactive yellow protein (PYP) from Halorhodospira halophila is a soluble 14 kDa blue-light photoreceptor. It absorbs light via its para-coumaric acid chromophore (pCA), which is covalently attached to Cys69 and is believed to be involved in the negative phototactic response of the organism to blue light. The complete structure (including H atoms) of PYP has been determined in D2O-soaked crystals through the application of joint X-ray (1.1Å) and neutron (2.5Å) structure refinement in combination with cross-validated maximum-likelihood simulated annealing. The resulting XN structure reveals that the phenolate O atom of pCA accepts deuterons from Glu46 Oε2 and Tyr42 Oη in two unusually short hydrogen bonds. This arrangement is stabilized by the donation of a deuteron from Thr50 Oγ1 to Tyr42 Oη. However, the deuteron position between pCA and Tyr42 is only partially occupied. Thus, this atom may also interact with Thr50, possibly being disordered or fluctuating between the two bonds.
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.
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.
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.
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.
Streptococcus agalactiae, a prokaryote that causes infections in neonates and immunocompromised adults, has a serine/threonine protein kinase (STK) signalling cascade. The structure of one of the targets, a family II inorganic pyrophosphatase, has been solved by molecular replacement and refined at 2.80 Å resolution to an R factor of 19.2% (Rfree = 26.7%). The two monomers in the asymmetric unit are related by a noncrystallographic twofold axis, but the biological dimer is formed by a crystallographic twofold. Each monomer contains the pyrophosphate analogue imidodiphosphate (PNP) and three metal ions per active site: two Mn2+ ions in sites M1 and M2 and an Mg2+ ion in site M3. The enzyme is in the closed conformation. Like other family II enzymes, the structure consists of two domains (residues 1–191 and 198–311), with the active site located between them. The conformation of Lys298 in the active site is different from those observed previously and it coordinates to the conserved DHH motif in a unique way. The structure suggests that Ser150, Ser194, Ser195 and Ser296 are the most likely targets for the Ser/Thr kinase and phosphatase because they are surface-accessible and either in the active site or in the hinge region between the two domains.
Nitroalkane oxidase (NAO), a flavoprotein cloned and purified from Fusarium oxysporum, catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones, with the production of H2O2 and nitrite. In this paper, the crystallization and preliminary X-ray data analysis of three crystal forms of active nitroalkane oxidase are described. The first crystal form belongs to a trigonal space group (either P3121 or P3221, with unit-cell parameters a = b = 103.8, c = 487.0 Å) and diffracts to at least 1.6 Å resolution. Several data sets were collected using 2θ and κ geometry in order to obtain a complete data set to 2.07 Å resolution. Solvent-content and Matthews coefficient analysis suggests that crystal form 1 contains two homotetramers per asymmetric unit. Crystal form 2 (P212121; a = 147.3, b = 153.5, c = 169.5 Å) and crystal form 3 (P31 or P32; a = b = 108.9, c = 342.5 Å) are obtained from slightly different conditions and also contain two homotetramers per asymmetric unit, but have different solvent contents. A three-wavelength MAD data set was collected from selenomethionine-enriched NAO (SeMet-NAO) in crystal form 3 and will be used for phasing.
A multi-subunit mitochondrial membrane protein complex involved in the Krebs Cycle and respiratory chain has been crystallized in a form suitable for near-atomic resolution structure determination.
A procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Å with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites.
Succinate dehydrogenase; respiration; protein structure; membrane protein; protein complex; respiratory enzyme; oxidoreductase; E.C. 184.108.40.206
The availability of high-intensity synchrotron facilities, technological advances in data-collection techniques and improved data-reduction and crystallographic software have ushered in a new era in high-throughput macromolecular crystallography. Here, the de novo automated crystal structure determination at 1.28 Å resolution of an NAD(P)H-dependent FMN reductase flavoprotein from Pseudomonas aeruginosa PA01-derived protein Q9I4D4 using the anomalous signal from an unusually small number of S atoms is reported. Although this protein lacks the flavodoxin key fingerprint motif [(T/S)XTGXT], it has been confirmed to bind flavin mononucleotide and the binding site was identified via X-ray crystallography. This protein contains a novel flavin mononucleotide-binding site GSLRSGSYN, which has not been previously reported. Detailed statistics pertaining to sulfur phasing and other factors contributing to structure determination are discussed. Structural comparisons of the apoenzyme and the protein complexed with flavin mononucleotide show conformational changes on cofactor binding. NADPH-dependent activity has been confirmed with biochemical assays.