Ab initio structure determination of nanocrystals of organic pharmaceutical compounds by electron diffraction at room temperature using a Timepix quantum area direct electron detector. Corrigendum.

Corrections are made to Table 1 in the article by van Genderen et al. [Acta Cryst. (2016), A72, 236-242].

Ab initio structure determination of nanocrystals of organic pharmaceutical compounds by electron diffraction at room temperature using a Timepix quantum area direct electron detector.

Until recently, structure determination by transmission electron microscopy of beam-sensitive three-dimensional nanocrystals required electron diffraction tomography data collection at liquid-nitrogen temperature, in order to reduce radiation damage. Here it is shown that the novel Timepix detector combines a high dynamic range with a very high signal-to-noise ratio and single-electron sensitivity, enabling ab initio phasing of beam-sensitive organic compounds. Low-dose electron diffraction data (∼ 0.013 e(-) Å(-2) s(-1)) were collected at room temperature with the rotation method. It was ascertained that the data were of sufficient quality for structure solution using direct methods using software developed for X-ray crystallography (XDS, SHELX) and for electron crystallography (ADT3D/PETS, SIR2014).

The prediction of postoperative stroke or death in patients with preoperative atrial fibrillation undergoing non-cardiac surgery: a VISION sub-study.

BACKGROUND: The optimal means of pre-operative risk stratification in patients with atrial fibrillation (AF) is uncertain. OBJECTIVE: To examine the accuracy of AF thromboembolic risk models (the CHADS2, CHA2DS2-VASc, and R2CHADS2 scores) for predicting 30-day stroke and/or all-cause mortality after non-cardiac surgery in patients with preoperative AF, and to compare these risk scores with the Revised Cardiac Risk Index (RCRI). PATIENTS/METHODS: A multicentre (8 countries, 2007-2011) prospective cohort study of patients ≥ 45 years of age undergoing inpatient non-cardiac surgery, who were followed until 30 days after surgery. We calculated c-statistics for each risk prediction model and net reclassification improvements (NRIs) compared with the RCRI. RESULTS: The 961 patients with preoperative AF were at higher risk of any cardiovascular event in the 30 days postoperatively compared with the 13 001 patients without AF: 26.6% vs. 9.0%; adjusted odds ratio, 1.58; 95% confidence interval [CI], 1.33-1.88. All thromboembolic risk scores predicted postoperative death just as well as the RCRI (with c-indices between 0.67 and 0.72). Compared with the RCRI (which had a c-index of 0.64 for 30-day stroke/death), the CHADS2 (c-index, 0.67; NRI, 0.31; 95% CI, 0.02-0.61) significantly improved postoperative stroke/mortality risk prediction, largely due to improved discrimination of patients who did not subsequently have an event. CONCLUSIONS: In AF patients, the three thromboembolic risk scores performed similarly to the RCRI in predicting death within 30 days and the CHADS2 score was the best predictor of postoperative stroke/death regardless of type of surgery.

Practice pattern variation in oral glucocorticoid therapy after the induction of response in proliferative lupus nephritis.

Glucocorticoids are standard therapy for induction of response in proliferative lupus nephritis. However, the optimal duration of glucocorticoid therapy is uncertain. We surveyed physicians who treat lupus nephritis regarding their use of glucocorticoids in proliferative lupus nephritis after induction of response and regarding factors associated with different practice patterns. We administered a questionnaire of standardized cases assessing glucocorticoid use after induction of response to specialists with expertise in proliferative lupus nephritis. We examined the association between continuation of glucocorticoids and patient and physician characteristics. Of 90 invited participants, 72 (80%) responded. A total of 24 (33%) respondents attempted to discontinue glucocorticoids in all scenarios, 21 (29%) continued glucocorticoids in all scenarios, and 27 (38%) attempted to discontinue in some scenarios but not others. Responses varied according to the physician group (p < 0.001) and by years in practice (p < 0.001). Of those who discontinued glucocorticoids in selected scenarios, 15/27 (55%) were influenced by the characteristics of the induction of response, 16/27 (59%) by past lupus history, and 9/27 (33%) by the tolerance and use of immunosuppression. We conclude that glucocorticoid therapy after induction of response in proliferative lupus nephritis is varied. This variability likely represents clinical equipoise. A randomized trial evaluating the effect of glucocorticoid use after induction of response is warranted.

Fluid and volume monitoring.

BACKGROUND: Fluid resuscitation is not only used to prevent acute kidney injury (AKI) but fluid management is also a cornerstone of treatment for patients with established AKI and renal failure. Ultrafiltration removes volume initially from the intravascular compartment inducing a relative degree of hypovolemia. Normal reflex mechanisms attempt to sustain blood pressure constant despite marked changes in blood volume and cardiac output. Thus, compensated shock with a normal blood pressure is a major cause of AKI or exacerbations of AKI during ultrafiltration. METHODS: We undertook a systematic review of the literature using MEDLINE, Google Scholar and PubMed searches. We determined a list of key questions and convened a 2-day consensus conference to develop summary statements via a series of alternating breakout and plenary sessions. In these sessions, we identified supporting evidence and generated clinical practice recommendations and/or directions for future research. RESULTS: We defined three aspects of fluid monitoring: i) normal and pathophysiological cardiovascular mechanisms; ii) measures of volume responsiveness and impending cardiovascular collapse during volume removal, and; iii) measured indices of each using non-invasive and minimally invasive continuous and intermittent monitoring techniques. The evidence documents that AKI can occur in the setting of normotensive hypovolemia and that under-resuscitation represents a major cause of both AKI and mortality ion critically ill patients. Traditional measures of intravascular volume and ventricular filling do not predict volume responsiveness whereas dynamic functional hemodynamic markers, such as pulse pressure or stroke volume variation during positive pressure breathing or mean flow changes with passive leg raising are highly predictive of volume responsiveness. Numerous commercially-available devices exist that can acquire these signals. CONCLUSIONS: Prospective clinical trials using functional hemodynamic markers in the diagnosis and management of AKI and volume status during ultrafiltration need to be performed. More traditional measure of preload be abandoned as marked of volume responsiveness though still useful to assess overall volume status.

A mutant Shiga-like toxin IIe bound to its receptor Gb(3): structure of a group II Shiga-like toxin with altered binding specificity.

BACKGROUND: Shiga-like toxins (SLTs) are produced by the pathogenic strains of Escherichia coli that cause hemorrhagic colitis and hemolytic uremic syndrome. These diseases in humans are generally associated with group II family members (SLT-II and SLT-IIc), whereas SLT-IIe (pig edema toxin) is central to edema disease of swine. The pentameric B-subunit component of the majority of family members binds to the cell-surface glycolipid globotriaosyl ceramide (Gb(3)), but globotetraosyl ceramide (Gb(4)) is the preferred receptor for SLT-IIe. A double-mutant of the SLT-IIe B subunit that reverses two sequence differences from SLT-II (GT3; Gln65-->Glu, Lys67-->Gln, SLT-I numbering) has been shown to bind more strongly to Gb(3) than to Gb(4). RESULTS: To understand the molecular basis of receptor binding and specificity, we have determined the structure of the GT3 mutant B pentamer, both in complex with a Gb(3) analogue (2.0 A resolution; R = 0.155, R(free) = 0.194) and in its native form (2.35 A resolution; R = 0.187, R(free) = 0.232). CONCLUSIONS: These are the first structures of a member of the medically important group II Shiga-like toxins to be reported. The structures confirm the previous observation of multiple binding sites on each SLT monomer, although binding site 3 is not occupied in the GT3 structure. Analysis of the binding properties of mutants suggests that site 3 is a secondary Gb(4)-binding site. The two mutated residues are located appropriately to interact with the extra betaGalNAc residue on Gb(4). Differences in the binding sites provide a molecular basis for understanding the tissue specificities and pathogenic mechanisms of members of the SLT family.

Shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands.

The diseases caused by Shiga and cholera toxins account for the loss of millions of lives each year. Both belong to the clinically significant subset of bacterial AB5 toxins consisting of an enzymatically active A subunit that gains entry to susceptible mammalian cells after oligosaccharide recognition by the B5 homopentamer. Therapies might target the obligatory oligosaccharide-toxin recognition event, but the low intrinsic affinity of carbohydrate-protein interactions hampers the development of low-molecular-weight inhibitors. The toxins circumvent low affinity by binding simultaneously to five or more cell-surface carbohydrates. Here we demonstrate the use of the crystal structure of the B5 subunit of Escherichia coli O157:H7 Shiga-like toxin I (SLT-I) in complex with an analogue of its carbohydrate receptor to design an oligovalent, water-soluble carbohydrate ligand (named STARFISH), with subnanomolar inhibitory activity. The in vitro inhibitory activity is 1-10-million-fold higher than that of univalent ligands and is by far the highest molar activity of any inhibitor yet reported for Shiga-like toxins I and II. Crystallography of the STARFISH/Shiga-like toxin I complex explains this activity. Two trisaccharide receptors at the tips of each of five spacer arms simultaneously engage all five B subunits of two toxin molecules.

A 2.6 A structure of a serpin polymer and implications for conformational disease.

The function of the serpins as proteinase inhibitors depends on their ability to insert the cleaved reactive centre loop as the fourth strand in the main A beta-sheet of the molecule upon proteolytic attack at the reactive centre, P1-P1'. This mechanism is vulnerable to mutations which result in inappropriate intra- or intermolecular loop insertion in the absence of cleavage. Intermolecular loop insertion is known as serpin polymerisation and results in a variety of diseases, most notably liver cirrhosis resulting from mutations of the prototypical serpin alpha1-antitrypsin. We present here the 2.6 A structure of a polymer of alpha1-antitrypsin cleaved six residues N-terminal to the reactive centre, P7-P6 (Phe352-Leu353). After self insertion of P14 to P7, intermolecular linkage is affected by insertion of the P6-P3 residues of one molecule into the partially occupied beta-sheet A of another. This results in an infinite, linear polymer which propagates in the crystal along a 2-fold screw axis. These findings provide a framework for understanding the uncleaved alpha1-antitrypsin polymer and fibrillar and amyloid deposition of proteins seen in other conformational diseases, with the ordered array of polymers in the crystal resulting from slow accretion of the cleaved serpin over the period of a year.

The active conformation of plasminogen activator inhibitor 1, a target for drugs to control fibrinolysis and cell adhesion.

BACKGROUND: Plasminogen activator inhibitor 1 (PAI-1) is a serpin that has a key role in the control of fibrinolysis through proteinase inhibition. PAI-1 also has a role in regulating cell adhesion processes relevant to tissue remodeling and metastasis; this role is mediated by its binding to the adhesive glycoprotein vitronectin rather than by proteinase inhibition. Active PAI-1 is metastable and spontaneously transforms to an inactive latent conformation. Previous attempts to crystallize the active conformation of PAI-1 have failed. RESULTS: The crystal structure of a stable quadruple mutant of PAI-1(Asn150-->His, Lys154-->Thr, Gln319-->Leu, Met354-->Ile) in its active conformation has been solved at a nominal 3 A resolution. In two of four independent molecules within the crystal, the flexible reactive center loop is unconstrained by crystal-packing contacts and is disordered. In the other two molecules, the reactive center loop forms intimate loop-sheet interactions with neighboring molecules, generating an infinite chain within the crystal. The overall conformation resembles that seen for other active inhibitory serpins. CONCLUSIONS: The structure clarifies the molecular basis of the stabilizing mutations and the reduced affinity of PAI-1, on cleavage or in the latent form, for vitronectin. The infinite chain of linked molecules also suggests a new mechanism for the serpin polymerization associated with certain diseases. The results support the concept that the reactive center loop of an active serpin is flexible and has no defined conformation in the absence of intermolecular contacts. The determination of the structure of the active form constitutes an essential step for the rational design of PAI-1 inhibitors.

Extending the limits of molecular replacement through combined simulated annealing and maximum-likelihood refinement.

Phases determined by the molecular-replacement method often suffer from model bias. In extreme cases, the refinement of the atomic model can stall at high free R values when the resulting electron-density maps provide little indication of how to correct the model, sometimes rendering even a correct solution unusable. Here, it is shown that several recent advances in refinement methodology allow productive refinement, even in cases where the molecular-replacement-phased electron-density maps do not allow manual rebuilding. In test calculations performed with a series of homologous models of penicillopepsin using either backbone atoms, or backbone atoms plus conserved core residues, model bias is reduced and refinement can proceed efficiently, even if the initial model is far from the correct one. These new methods combine cross-validation, torsion-angle dynamics simulated annealing and maximum-likelihood target functions. It is also shown that the free R value is an excellent indicator of model quality after refinement, potentially discriminating between correct and incorrect molecular-replacement solutions. The use of phase information, even in the form of bimodal single-isomorphous-replacement phase distributions, greatly improves the radius of convergence of refinement and hence the quality of the electron-density maps, further extending the limits of molecular replacement.

Crystallography & NMR system: A new software suite for macromolecular structure determination.

A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.

Incorporation of prior phase information strengthens maximum-likelihood structure refinement.

The application of a maximum-likelihood analysis to the problem of structure refinement has led to striking improvements over the traditional least-squares methods. Since the method of maximum likelihood allows for a rational incorporation of other sources of information, we have derived a likelihood function that incorporates experimentally determined phase information. In a number of different test cases, this target function performs better than either a least-squares target or a maximum-likelihood function lacking prior phases. Furthermore, this target gives significantly better results compared with other functions incorporating phase information. When combined with a procedure to mask 'unexplained' density, the phased likelihood target also makes it possible to refine very incomplete models.

Cross-validated maximum likelihood enhances crystallographic simulated annealing refinement.

Recently, the target function for crystallographic refinement has been improved through a maximum likelihood analysis, which makes proper allowance for the effects of data quality, model errors, and incompleteness. The maximum likelihood target reduces the significance of false local minima during the refinement process, but it does not completely eliminate them, necessitating the use of stochastic optimization methods such as simulated annealing for poor initial models. It is shown that the combination of maximum likelihood with cross-validation, which reduces overfitting, and simulated annealing by torsion angle molecular dynamics, which simplifies the conformational search problem, results in a major improvement of the radius of convergence of refinement and the accuracy of the refined structure. Torsion angle molecular dynamics and the maximum likelihood target function interact synergistically, the combination of both methods being significantly more powerful than each method individually. This is demonstrated in realistic test cases at two typical minimum Bragg spacings (dmin = 2.0 and 2.8 A, respectively), illustrating the broad applicability of the combined method. In an application to the refinement of a new crystal structure, the combined method automatically corrected a mistraced loop in a poor initial model, moving the backbone by 4 A.