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The experimental charge density of a highly substituted fullerene derivative, the T(sub h)-symmetrical dodekakis-(ethoxycarbonyl)-C60-fullerene cocristallized with difluorobenzene, was determined. This state-of-the-artstudy confirms that presently available technical standards allow for accurate experimental charge density determinations on the structures containing more than 100 atoms.

Strategies for collecting X‐ray diffraction data have evolved alongside beamline hardware and detector developments. The traditional approaches for diffraction data collection have emphasised collecting data from noisy integrating detectors (i.e. film, image plates and CCD detectors). With fast pixel array detectors on stable beamlines, the limiting factor becomes the sample lifetime, and the question becomes one of how to expend the photons that your sample can diffract, i.e. as a smaller number of stronger measurements or a larger number of weaker data. This parameter space is explored via experiment and synthetic data treatment and advice is derived on how best to use the equipment on a modern beamline. Suggestions are also made on how to acquire data in a conservative manner if very little is known about the sample lifetime. Different modes of data collection are explored, and the effect of flux and multiplicity on the resulting quality of the data set is discussed. Advice is offered on how to collect data in the absence of prior knowledge of the sample. [ABSTRACT FROM AUTHOR]

Amino acid structures are an ideal test set for method-development studies in crystallography. High-resolution X-ray diffraction data for eight previously studied genetically encoding amino acids are provided, complemented by a non-standard amino acid. Structures were re-investigated to study a widely applicable treatment that permits accurate X−H bond lengths to hydrogen atoms to be obtained: this treatment combines refinement of positional hydrogen-atom parameters with aspherical scattering factors with constrained 'TLS+INV' estimated hydrogen anisotropic displacement parameters (H-ADPs). Tabulated invariom scattering factors allow rapid modeling without further computations, and unconstrained Hirshfeld atom refinement provides a computationally demanding alternative when database entries are missing. Both should incorporate estimated H-ADPs, as free refinement frequently leads to over-parameterization and non-positive definite H-ADPs irrespective of the aspherical scattering model used. Using estimated H-ADPs, both methods yield accurate and precise X−H distances in best quantitative agreement with neutron diffraction data (available for five of the test-set molecules). This work thus solves the last remaining problem to obtain such results more frequently. Density functional theoretical QM/MM computations are able to play the role of an alternative benchmark to neutron diffraction. [ABSTRACT FROM AUTHOR]

Gastrointestinal bacteria, like Escherichia coli, must remove bile acid to survive in the gut. Bile acid removal in E. coli is thought to be mediated primarily by the multidrug efflux pump, AcrB. Here, we present the structure of E. coli AcrB in complex with deoxycholate at 3.85 Å resolution. All evidence suggests that bile acid is transported out of the cell via the periplasmic vestibule of the AcrAB-TolC complex. [ABSTRACT FROM AUTHOR]

The estrogen-related receptor (ERR) γ behaves as a constitutive activator of transcription. Although no natural ligand is known, ERRγ is deactivated by the estrogen receptor (ER) agonist diethylstilbestrol and the selective ER modulator 4-hydroxytamoxifen but does not significantly respond to estradiol or raloxifene. Here we report the crystal structures of the ERRγ ligand binding domain (LBD) complexed with diethylstilbestrol or 4-hydroxytamoxifen. Antagonist binding to ERRT results in a rotation of the side chain of Phe-435 that partially fills the cavity of the apoLBD. The new rotamer of Phe-435 displaces the ‘activation helix’ (helix 12) from the agonist position observed in the absence of ligand. In contrast to the complexes of the ERα LBD with 4-hydroxytamoxifen or raloxifene, helix 12 of antagonist-bound ERRγ does not occupy the coactivator groove but appears to be completely dissociated from the LBD body. Comparison of the ligand-bound LBDs of ERRγ and ERα reveals small but significant differences in the architecture of the ligand binding pockets that result in a slightly shifted binding position of diethylstilbestrol and a small rotation of 4-hydroxytamoxifen in the cavity of ERRγ relative to ERα. Our results provide detailed molecular insight into the conformational changes occurring upon binding of synthetic antagonists to the constitutive orphan receptor ERRγ and reveal structural differences with ERs that explain why ERRγ does not bind estradiol or raloxifene and will help to design new selective antagonists. [ABSTRACT FROM AUTHOR]

Retinoids have demonstrated therapeutic efficacy in the treatment of acute promyelocytic leukaemia and in the chemoprevention of a large number of cancers. As the cellular signalling pathway of retinoids can be transduced by the three retinoic acid receptor (RAR) isotypes α, β and γ, the side effects of these treatments induced efforts to generate isotype-selective ligands. Despite knowledge of the crystal structures of RARα and RARγ ligand-binding domains (LBDs), the rational design of such ligands has been hampered by the absence of RARβ LBD structural data. Here, a strategy used to express a large-scale soluble fraction of the human RARβ LBD suitable for biophysical analysis is reported, as well as a procedure for crystallizing it bound to a synthetic retinoid (TTNPB) with or without a co-activator peptide (SRC-1). Preliminary X-ray analysis revealed that both complexes crystallized in the orthorhombic space group P212121. The unit-cell parameters are a = 47.81, b = 58.52, c = 92.83 Å for the TTNPB-hRARβ LBD crystal and a = 58.14, b = 84.07, c = 102.37 Å when the SRC-1 peptide is also bound. [ABSTRACT FROM AUTHOR]

Although structural studies on the ligand-binding domain (LBD) have established the general mode of nuclear receptor (NR)/coactivator interaction, determinants of binding specificity are only partially understood. The LBD of estrogen receptor-α (ERα), for example, interacts only with a region of peroxisome proliferator-activated receptor coactivator (PGC)-1α, which contains the canonical LXKLL motif (NR box2), whereas the LBD of estrogen-related receptor-α (ERRα) also binds efficiently an untypical, LXXYL-containing region (NR box3) of PGC-1α. Surprisingly, in a previous structural study, the ERα LBD has been observed to bind NR box3 of transcriptional intermediary factor (TIF)-2 untypically via LXXYL, whereas the ERRα LBD binds this region of TIF-2 only poorly. Here we present a new crystal structure of the ERRα LBD in complex with a PGC-1α box3 peptide. In this structure, residues N-terminal of the PGC-1α LXXYL motif formed contacts with helix 4, the loop connecting helices 8 and 9, and with the C terminus of the ERRα LBD. Interaction studies using wild-type and mutant PGC-1α and ERRα showed that these contacts are functionally relevant and are required for efficient ERRα/PGC-1α interaction. Furthermore, a structure comparison between ERRα and ERα and mutation analyses provided evidence that the helix 8-9 loop, which differs significantly in both nuclear receptors, is a major determinant of coactivator binding specificity. Finally, our results revealed that in ERRα the helix 8-9 loop allosterically links the LBD homodimer interface with the coactivator cleft, thus providing a plausible explanation for distinct PGC-1α binding to ERRα monomers and homodimers. [ABSTRACT FROM AUTHOR]

Amino acid structures are an ideal test set for method-development studies in crystallography. High-resolution X-ray diffraction data for eight previously studied genetically encoding amino acids are provided, complemented by a non-standard amino acid. Structures were re-investigated to study a widely applicable treatment that permits accurate X-H bond lengths to hydrogen atoms to be obtained: this treatment combines refinement of positional hydrogen-atom parameters with aspherical scattering factors with constrained "TLS+INV" estimated hydrogen anisotropic displacement parameters (H-ADPs). Tabulated invariom scattering factors allow rapid modeling without further computations, and unconstrained Hirshfeld atom refinement provides a computationally demanding alternative when database entries are missing. Both should incorporate estimated H-ADPs, as free refinement frequently leads to over-parameterization and non-positive definite H-ADPs irrespective of the aspherical scattering model used. Using estimated H-ADPs, both methods yield accurate and precise X-H distances in best quantitative agreement with neutron diffraction data (available for five of the test-set molecules). This work thus solves the last remaining problem to obtain such results more frequently. Density functional theoretical QM/MM computations are able to play the role of an alternative benchmark to neutron diffraction., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Prostate cancer evolution is driven by a combination of epigenetic and genetic alterations such as coordinated chromosomal rearrangements, termed chromoplexy. TMPRSS2-ERG gene fusions found in human prostate tumors are a hallmark of chromoplexy. TMPRSS2-ERG fusions have been linked to androgen signaling and depend on androgen receptor (AR)-coupled gene transcription. Here, we show that dimethylation of KDM1A at K114 (to form K114me2) by the histone methyltransferase EHMT2 is a key event controlling androgen-dependent gene transcription and TMPRSS2-ERG fusion. We identified CHD1 as a KDM1A K114me2 reader and characterized the KDM1A K114me2-CHD1 recognition mode by solving the cocrystal structure. Genome-wide analyses revealed chromatin colocalization of KDM1A K114me2, CHD1 and AR in prostate tumor cells. Together, our data link the assembly of methylated KDM1A and CHD1 with AR-dependent transcription and genomic translocations, thereby providing mechanistic insight into the formation of TMPRSS2-ERG gene fusions during prostate-tumor evolution.

Macromolecular crystallography (MX) is the most powerful technique available to structural biologists to visualize in atomic detail the macromolecular machinery of the cell. Since the emergence of structural genomics initiatives, significant advances have been made in all key steps of the structure determination process. In particular, third-generation synchrotron sources and the application of highly automated approaches to data acquisition and analysis at these facilities have been the major factors in the rate of increase of macromolecular structures determined annually. A plethora of tools are now available to users of synchrotron beamlines to enable rapid and efficient evaluation of samples, collection of the best data, and in favorable cases structure solution in near real time. Here, we provide a short overview of the emerging use of collecting X-ray diffraction data directly from the crystallization experiment. These in situ experiments are now routinely available to users at a number of synchrotron MX beamlines. A practical guide to the use of the method on the MX suite of beamlines at Diamond Light Source is given.

NGFI-B is a ligand-independent orphan nuclear receptor of the NR4A subfamily that displays important functional differences with its homolog Nurr1. In particular, the NGFI-B ligand-binding domain (LBD) exhibits only modest activity in cell lines in which the Nurr1 LBD strongly activates transcription. To gain insight into the structural basis for the distinct activation potentials, we determined the crystal structure of the NGFI-B LBD at 2.4-angstroms resolution. Superimposition with the Nurr1 LBD revealed a significant shift of the position of helix 12, potentially caused by conservative amino acids exchanges in helix 3 or helix 12. Replacement of the helix 11-12 region of Nurr1 with that of NGFI-B dramatically reduces the transcriptional activity of the Nurr1 LBD. Similarly, mutation of Met414 in helix 3 to leucine or of Leu591 in helix 12 to isoleucine (the corresponding residues found in NGFI-B) significantly affects Nurr1 transactivation. In comparison, swapping the helix 11-12 region of Nurr1 into NGFI-B results in a modest increase of activity. These observations reveal a high sensitivity of LBD activity to changes that influence helix 12 positioning. Furthermore, mutation of hydrophobic surface residues in the helix 11-12 region (outside the canonical co-activator surface constituted by helices 3, 4, and 12) severely affects Nurr1 transactivation. Together, our data suggest that a novel co-regulator surface that includes helix 11 and a specifically positioned helix 12 determine the cell type-dependent activities of the NGFI-B and the Nurr1 LBD.

Two penicillin derivatives, the active penamecillin and the inactive penamecillin-1beta-sulfoxide, were used to study the relationship between their charge density and their activity. Single crystals of both compounds were measured at the synchrotron beamline F1 at the HASYLAB/DESY, at 100 K and up to resolutions of around 0.4 A. Experimental charge densities were obtained by using the Hansen-Coppens multipole formalism. The cleavage of the amide bond in the beta-lactam ring is of paramount importance in the mechanism of action of penicillins. Topological analysis of this bond in terms of Bader's AIM theory showed that its strength is equal in both compounds; therefore a direct influence of bond strength on the activity can be ruled out. However, the two derivatives differ significantly in their experimental electrostatic potentials. These differences are discussed and provide further insight into the chemistry and activity of penicillins.

Synchrotron radiation and CCD detection give the possibility for fast diffraction experiments, which were employed to deduce the exact charge-density distributions of some amino acids. Their topological analysis (the figure shows the negative Laplacian function of dl-serine in the plane of the carboxylate group) yields not only comparable information about intramolecular but also about weak intermolecular interactions., (© 1999 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.)