DiffraGAN: a conditional generative adversarial network for phasing single molecule diffraction data to atomic resolution.

Introduction: Proteins that adopt multiple conformations pose significant challenges in structural biology research and pharmaceutical development, as structure determination via single particle cryo-electron microscopy (cryo-EM) is often impeded by data heterogeneity. In this context, the enhanced signal-to-noise ratio of single molecule cryo-electron diffraction (simED) offers a promising alternative. However, a significant challenge in diffraction methods is the loss of phase information, which is crucial for accurate structure determination. Methods: Here, we present DiffraGAN, a conditional generative adversarial network (cGAN) that estimates the missing phases at high resolution from a combination of single particle high-resolution diffraction data and low-resolution image data. Results: For simulated datasets, DiffraGAN allows effectively determining protein structures at atomic resolution from diffraction patterns and noisy low-resolution images. Discussion: Our findings suggest that combining single particle cryo-electron diffraction with advanced generative modeling, as in DiffraGAN, could revolutionize the way protein structures are determined, offering an alternative and complementary approach to existing methods.

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].

New developments in phase refinement.

A longstanding problem in X-ray crystallography is that vital information regarding the crystal phases in missing from the experimental data that are gathered in the diffraction experiment. Prior knowledge needs to be introduced in order to resolve phase ambiguities whenever the diffraction data are not sufficient to unequivocally reconstruct the crystal phases through anomalous or isomorphous differences. Very recent developments include progress in the application of direct methods to small proteins and other compounds of a similar small size (Shake 'n' Bake, SHELXD, CRUNCH and SIR96), bias-free refinement through the gamma-correction (Solomon), improvements in the determination of phase probability distributions (SHARP) and automated atomic refinement (wARP).

Protein structure determination by electron diffraction using a single three-dimensional nanocrystal.

Three-dimensional nanometre-sized crystals of macromolecules currently resist structure elucidation by single-crystal X-ray crystallography. Here, a single nanocrystal with a diffracting volume of only 0.14 µm3, i.e. no more than 6 × 105 unit cells, provided sufficient information to determine the structure of a rare dimeric polymorph of hen egg-white lysozyme by electron crystallography. This is at least an order of magnitude smaller than was previously possible. The molecular-replacement solution, based on a monomeric polyalanine model, provided sufficient phasing power to show side-chain density, and automated model building was used to reconstruct the side chains. Diffraction data were acquired using the rotation method with parallel beam diffraction on a Titan Krios transmission electron microscope equipped with a novel in-house-designed 1024 × 1024 pixel Timepix hybrid pixel detector for low-dose diffraction data collection. Favourable detector characteristics include the ability to accurately discriminate single high-energy electrons from X-rays and count them, fast readout to finely sample reciprocal space and a high dynamic range. This work, together with other recent milestones, suggests that electron crystallography can provide an attractive alternative in determining biological structures.

The impact of single amino acid substitutions in CD3gamma on the CD3epsilongamma interaction and T-cell receptor-CD3 complex formation.

The human T-cell receptor-CD3 complex consists of at least eight polypeptide chains; CD3gamma- and delta-dimers associate with the disulfide linked alphabeta- and zetazeta-dimers to form a functional receptor complex. The exact structure of this complex is still unknown. We now have examined the interaction between CD3gamma and CD3 in human T-cells. For this purpose, we have generated site-directed mutants of CD3gamma that were introduced in human T-cells defective in CD3gamma expression. Cell-surface and intracellular expression of the introduced CD3gamma chains was determined, as was the association with CD3delta, CD3, and the T-cell receptor. Although the introduction of wild type CD3gamma and CD3gamma (78Y-F) fully restored T-cell receptor assembly and expression, the introduction of CD3gamma (82C-S), CD3gamma (85C-S), and CD3gamma (76Q-E) all resulted in an impaired association between CD3gamma and CD3 and a lack of cell-surface expressed CD3gamma. Finally, the introduction of CD3gamma (76Q-L) and CD3gamma (78Y-A) restored the expression of TCR-CD3deltagammazeta2 complexes, although the association between CD3gamma and CD3 was impaired. These results indicate that several amino acids in CD3gamma are essential for an optimal association between CD3gamma and CD3 and the assembly of a cell-surface expressed TCR-CD3deltagammazeta2 complex.

The crystal structure of the nucleotide-free alpha 3 beta 3 subcomplex of F1-ATPase from the thermophilic Bacillus PS3 is a symmetric trimer.

BACKGROUND: F1-ATPase, an oligomeric assembly with subunit stoichiometry alpha 3 beta 3 gamma delta epsilon, is the catalytic component of the ATP synthase complex, which plays a central role in energy transduction in bacteria, chloroplasts and mitochondria. The crystal structure of bovine mitochondrial F1-ATPase displays a marked asymmetry in the conformation and nucleotide content of the catalytic beta subunits. The alpha 3 beta 3 subcomplex of F1-ATPase has been assembled from subunits of the moderately thermophilic Bacillus PS3 made in Escherichia coli, and the subcomplex is active but does not show the catalytic cooperativity of intact F1-ATPase. The structure of this subcomplex should provide new information on the conformational variability of F1-ATPase and may provide insights into the unusual catalytic mechanism employed by this enzyme. RESULTS: The crystal structure of the nucleotide-free bacterial alpha 3 beta 3 subcomplex of F1-ATPase, determined at 3.2 A resolution, shows that the oligomer has exact threefold symmetry. The bacterial beta subunits adopt a conformation essentially identical to that of the nucleotide-free beta subunit in mitochondrial F1-ATPase; the alpha subunits have similar conformations in both structures. CONCLUSIONS: The structures of the bacterial F1-ATPase alpha and beta subunits are very similar to their counterparts in the mitochondrial enzyme, suggesting a common catalytic mechanism. The study presented here allows an analysis of the different conformations adopted by the alpha and beta subunits and may ultimately further our understanding of this mechanism.

Lattice filter for processing image data of three-dimensional protein nanocrystals.

When 300 kV cryo-EM images at Scherzer focus are acquired from ∼ 100 nm thick three-dimensional protein nanocrystals using a Falcon 2 direct electron detector, Fourier transformation can reveal the crystalline lattice to surprisingly high resolutions, even though the images themselves seem to be devoid of any contrast. Here, it is reported how this lattice information can be enhanced by means of a wave finder in combination with Wiener-type maximum-likelihood filtering. This procedure paves the way towards full three-dimensional structure determination at high resolution for protein crystals.

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 interaction between aminoacyl-tRNA and the mutant elongation factors Tu AR and B0.

The binding of Tyr-[AEDANS-s2C]tRNA(Tyr) (Tyr-tRNA(Tyr) modified at the penultimate cytidine residue with a thio group at position 2 of the pyrimidine ring, to which an N-(acetylaminoethyl)-5-naphthylamine-1-sulfonic acid fluorescence group is attached) to mutant elongation factor (EF)-Tu species from E. coli, EF-TuAR (Ala-375----Thr) and EF-TuBO (Gly-222----Asp), both complexed to GTP, was investigated in absence of kirromycin by measuring the change in fluorescence of the modified tRNA induced by complex formation. The calculated dissociation constant in the case of EF-TuAR is about 4 nM and in the case of EF-TuB0, about 1 nM. These values are higher than that of wild-type EF-Tu, which was 0.24 nM measured with the same system. The affinity between either EF-TuB0.kirromycin.GDP or EF-TuB0.kirromycin.GTP on the one hand, and a mixture of aminoacyl-tRNAs on the other, was measured with zone-interference gel electrophoresis. The dissociation constants are 20 microM and 7 microM, respectively, a factor of about two higher than in the case of wild-type EF-Tu.kirromycin. These findings provide a clue for the observed increase in translational errors in strains carrying the mutations. Furthermore, the experiments with EF-TuB0.kirromycin deepen our understanding of the effects of the B0 mutation on the kirromycin phenotype of the mutant cells concerned.

Wild-type alpha 1-antitrypsin is in the canonical inhibitory conformation.

alpha 1-Antitrypsin is the archetypal member of the serine proteinase inhibitor or serpin superfamily. Members of the family show structural homology based on a dominant A beta-sheet and a mobile reactive centre loop. Our recent crystal structure of alpha 1-antitrypsin stabilized with a point mutation showed the loop to be in a canonical inhibitory conformation in the absence of significant insertion into the A beta-sheet. It could be argued that the stabilizing mutation may induce the reactive centre loop to adopt an artificial, and unrepresentative, conformation and the finding seems to be at variance with studies assessing rates of peptide insertion into the A beta-sheet and limited proteolysis of the reactive loop. Here we present a 2.9 A structure of recombinant wild-type alpha 1-antitrypsin with no stabilizing mutations. Again, the reactive loop is in a canonical conformation in the absence of significant insertion into the A beta-sheet. A stabilizing salt bridge between P5 glutamate and arginine residues 196, 223 and 281, already identified in the mutant, provides strong evidence that this conformation is not an artefact of crystallization but represents the conformation of the circulating inhibitor in vivo. Comparison with the structure of alpha 1-antitrypsin stabilized with the Phe51Leu mutation indicates that the increased thermal stability of the mutant results from enhanced packing of aromatic residues in the hydrophobic core of the molecule. The structure of wild-type alpha 1-antitrypsin reveals a hydrophobic pocket between s2A and helices D and E that is filled on reactive loop insertion and the formation of biologically relevant loop-sheet polymers. This pocket may provide a target for rational drug design to prevent the formation of polymers and the associated plasma deficiency, liver cirrhosis and emphysema.

The 2.6 A structure of antithrombin indicates a conformational change at the heparin binding site.

The crystal structure of a dimeric form of intact antithrombin has been solved to 2.6 A, representing the highest-resolution structure of an active, inhibitory serpin to date. The crystals were grown under microgravity conditions on Space Shuttle mission STS-67. The overall confidence in the structure, determined earlier from lower resolution data, is increased and new insights into the structure-function relationship are gained. Clear and continuous electron density is present for the reactive centre loop region P12 to P14 inserting into the top of the A-beta-sheet. Areas of the extended amino terminus, unique to antithrombin and important in the binding of the glycosaminoglycan heparin, can now be traced further than in the earlier structures. As in the earlier studies, the crystals contain one active and one latent molecule per asymmetric unit. Better definition of the electron density surrounding the D-helix and of the residues implicated in the binding of the heparin pentasaccharide (Arg47, Lys114, Lys125, Arg129) provides an insight into the change of affinity of binding that accompanies the change in conformation. In particular, the observed hydrogen bonding of these residues to the body of the molecule in the latent form explains the mechanism for the release of newly formed antithrombin-protease complexes into the circulation for catabolic removal.

Crystallization and preliminary X-ray diffraction analysis of two conformations of intact human antithrombin.

Human antithrombin has been crystallized by microdialysis at pH 6.7 using 18% (w/v) polyethylene glycol-4000 as precipitant. Under these conditions two crystal forms grew. The first started growing after ten days, diffracted to 3.0 A resolution and belongs to the monoclinic space group P2(1) with two molecules in the asymmetric unit and unit cell dimensions a = 70.1 A, b = 101.5 A, c = 90.5 A and beta = 105.9 A. The other crystal form took more than three months to appear, diffracted to 5.5 A and belongs to the hexagonal space group of either P6(1) or P6(5) with unit cell dimensions of a = b = 99.3 A and c = 152.9 A and two molecules in the asymmetric unit. The antithrombin redissolved from the monoclinic crystals was shown both by SDS-polyacrylamide gel electrophoresis and by protein sequence analysis to be intact while that from the hexagonal crystals was cleaved in the reactive centre loop between the P'2 and P'3 (i.e. Leu-Asn) residues. Further analysis of the intact inhibitor from the monoclinic crystals indicated that the antithrombin was present in two different conformations; an active form which could inhibit thrombin and form a stable complex with the protease, and a form which was inactive as an inhibitor and which also did not act as a substrate for thrombin. This latter form also had a low affinity for heparin and in these ways resembles latent antithrombin. The active material from the monoclinic crystals had an association rate constant with thrombin in the presence of heparin (kass) of 7.5 x 10(7) M-1 s-1 (kass for native antithrombin = 8.2 (+/- 1.0) x 10(7) M-1 s-1) indicating it still had effective heparin cofactor activity. X-ray diffraction analysis also suggests that two different protein conformations exist within the monoclinic crystals. Whereas the rotation function peak heights are equal for both molecules in the asymmetric unit using the structure of intact ovalbumin as a search model, one of the two molecules gives a much clearer signal than the other when the structures of the two cleaved serpins, alpha 1-antitrypsin and alpha 1-antichymotrypsin are used.

Crystal structure of the C-terminal SH2 domain of the p85alpha regulatory subunit of phosphoinositide 3-kinase: an SH2 domain mimicking its own substrate.

The binding properties of Src homology-2 (SH2) domains to phosphotyrosine (pY)-containing peptides have been studied in recent years with the elucidation of a large number of crystal and solution structures. Taken together, these structures suggest a general mode of binding of pY-containing peptides, explain the specificities of different SH2 domains, and may be used to design inhibitors of pY binding by SH2 domain-containing proteins. We now report the crystal structure to 1.8 A resolution of the C-terminal SH2 domain (C-SH2) of the P85alpha regulatory subunit of phosphoinositide 3-kinase (PI3 K). Surprisingly, the carboxylate group of Asp2 from a neighbouring molecule occupies the phosphotyrosine binding site and interacts with Arg18 (alphaA2) and Arg36 (betaB5), in a similar manner to the phosphotyrosine-protein interactions seen in structures of other SH2 domains complexed with pY peptides. It is the first example of a non-phosphate-containing, non-aromatic mimetic of phosphotyrosine binding to SH2 domains, and this could have implications for the design of substrate analogues and inhibitors. Overall, the crystal structure closely resembles the solution structure, but a number of loops which demonstrate mobility in solution are well defined by the crystal packing. C-SH2 has adopted a binding conformation reminiscent of the ligand bound N-terminal SH2 domain of PI3K, apparently induced by the substrate mimicking of a neighbouring molecule in the crystal.

Implications for function and therapy of a 2.9 A structure of binary-complexed antithrombin.

The crystal structure of a binary complex of human antithrombin with a peptide of the same sequence as its reactive loop (P14-P3) has been determined at 2.9 A. The peptide binds as the middle strand s4A in the A beta-sheet, homologously to that of the reactive loop in the latent and cleaved forms of antithrombin. Peptide binding results in the complete expulsion of the hinge region of the loop from the A beta-sheet although the conformation differs from that of heparin-activated antithrombin. The 36-fold increase in the rate of reaction of the binary complex with factor Xa indicates that full loop expulsion alone is not sufficient for complete heparin activation of antithrombin but that this is also dependent on the overall conformation of the molecule. Previous studies have demonstrated that reactive loop peptides can block or reverse the polymerisation of serpins associated with cirrhosis and thrombosis. The antithrombin binary complex structure defines the precise localisation of the blocking peptide in a serpin and provides the basis for rational drug design for mimetics that will prevent polymerisation in vivo and so ameliorate the associated disease.

Crystallization of F1-ATPase from bovine heart mitochondria.

Crystals of the F1-ATPase sector of the ATP synthase complex from bovine heart mitochondria have been grown from solutions containing polyethylene glycol 6000. The crystals diffract to 2.9 A resolution on a laboratory X-ray source. They are orthorhombic and belong to the space group P2(1)2(1)2(1). The unit cell axes are a = 285 A, b = 108 A, c = 140 A. There is one molecule of F1-ATPase in the asymmetric unit.

Apoptin's functional N- and C-termini independently bind DNA.

Apoptin induces apoptosis specifically in tumour cells, where Apoptin is enriched in the DNA-dense heterochromatin and nucleoli. In vitro, Apoptin interacts with dsDNA, forming large nucleoprotein superstructures likely to be relevant for apoptosis induction. Its N- and C-terminal domains also have cell-killing activity, although they are less potent than the full-length protein. Here, we report that both Apoptin's N- and C-terminal halves separately bound DNA, indicating multiple independent binding sites. The reduced cell killing activity of both truncation mutants was mirrored in vitro by a reduced affinity compared to full-length Apoptin. However, none of the truncation mutants cooperatively bound DNA or formed superstructures, which suggests that cooperative DNA binding by Apoptin is required for the formation of nucleoprotein superstructures. As Apoptin's N- and C-terminal fragments not only share apoptotic activity, but also affinity for DNA, we propose that both properties are functionally linked.

The influence of tRNA located at the P-site on the turnover of EF-Tu.GTP on ribosomes.

The turnover of EF-Tu.GTP on poly-U programmed ribosomes was measured both in the presence and in the absence of N-acetylated Phe-tRNA(Phe) at the P-site. The reaction was uncoupled from protein synthesis by omitting Phe-tRNA(Phe) at the A-site. In this reaction, the ribosome can be considered as an enzyme catalysing the transition of EF-Tu.GTP to EF-Tu.GTP. A constant EF-Tu.GTP concentration is maintained by regenerating GDP to GTP at the expense of phosphoenolpyruvate by pyruvate kinase. The rate constants are determined using a procedure which corrects for the reduction in specific activity of GTP due to regeneration of the nucleotide. Ribosomes with an occupied P-site are more efficient in stimulating the GTPase of EF-Tu.GTP than ribosomes with an empty P-site. The data suggest that this is mainly caused by an increased affinity of EF-Tu.GTP for ribosomes with a filled P-site rather than by an enhanced reactivity of the GTPase centre.

Behavioral improvement in long-term geriatric patients during an age-integrated psychosocial rehabilitation program.

A study was made of the effects of a psychosocial rehabilitative program on the behavioral functioning of elderly chronically ill patients. High school students served as remotivation and socialization therapists in a supervised structured process designed to improve the quality of life for the participating nursing-home residents. The participants were 12 long-term patients whose ages ranged from 62 to 99 years (mean, 73.2 years). The effectiveness of the program was evaluated by means of the Sickness Impact Profile (SIP), a questionnaire designed to assess the effect of a physical illness on daily activities, psychosocial skills and mental status. The results demonstrated that the rehabilitative program had a significant impact on several dimensions of the lives of the participants. As a consequence of the interaction with the students, there was an increase in social interaction, a reduction in daytime sleeping and an increase in mobility. The results reported here extend the successful use of remotivation techniques to areas of overt behavioral functioning not previously assessed.

Relationship of age and biomedical risk factors to progression of coronary artery disease.

The relationship between age, biomedical risk factors and the progression of occlusive disease of the coronary arteries was studied in 176 patients (age range, 27-66 years) who had undergone at least two cine angiograms. The biomedical risk factors of interest were serum concentrations of cholesterol and triglycerides, smoking, hypertension, diabetes mellitus, family history of coronary disease, electrocardiographic abnormalities, obesity, and age. The findings did not reveal any significant differences in mean lipid levels between patients showing progression of disease and those who did not. However, the distribution of serum cholesterol values indicated more hypercholesterolemic patients among the disease-progression group, and more patients with ideal cholesterol levels among the no-progression group. The other biomedical variables did not appear to be related to the progression of coronary disease. Among the older patients, hypercholesterolemia and diabetes mellitus were related to disease progression. Among the younger patients, smoking was related to progression.