Enzyme intermediates captured on the fly by mix-and-inject serial crystallography

Background Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Results Here, we demonstrate a general method for capturing enzyme catalysis in action by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2s. Conclusions MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.This work was supported by the National Science Foundation (NSF)-Science and Technology Center (STC) BioXFEL through award STC-1231306, and in part by the US Department of Energy, Office of Science, Basic Energy Sciences under contract DE-SC0002164 (to A.O., algorithm design and development) and by the NSF under contract number 1551489 (to A.O., underlying analytical models). Portions of this research were performed at the Linac Coherent Light Source (LCLS). Use of the LCLS, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Basic Energy Sciences under contract DE-AC02-76SF00515. This material is based upon work supported by the NSF Graduate Research Fellowship Program to J.L.O. under grant no. 1450681. The work was also supported by funds from the National Institutes of Health grants R01 GM117342-01 and R01 GM095583, by funds from the Biodesign Center for Applied Structural Discovery at Arizona State University, and the US Department of Energy through Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344. Part of this work was also supported by program-oriented funds of the Helmholtz Association

Fibre FISH using BACs that bound the chirality locus.

<p>The gap between the hybridisation signal from BAC clones containing loci rad4 (red, R6F, 129 kb) and rad5 (green, B2D, 97 kb) is at least 4 to 5 times the length of a BAC clone. The preparation is counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). In this example from a genetically dextral snail, an interphase, meiotic cell is also visible, showing signal from hybridisation to both BACs.</p

Pachytene-FISH mapping of the chirality locus.

<p>Pachytene-FISH was performed using BACs containing RAD-Seq locus rad7 (white; BAC clone G1A, 137 kb), rad4 (red; R6F, 129 kb) and rad5 (green; B2D, 97 kb). All three BACs show hybridisation to the same bivalent chromosome. Chromosomes are counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue).</p

Summary of genotypes of all individuals.

<p>Summary of genotypes of all individuals.</p

Protein structure determination by single-wavelength anomalous diffraction phasing of X-ray free-electron laser data

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems that are prone to radiation damage. However, phasing XFEL data de novo is complicated by the inherent inaccuracy of SFX data, and only a few successful examples, mostly based on exceedingly strong anomalous or isomorphous difference signals, have been reported. Here, it is shown that SFX data from thaumatin microcrystals can be successfully phased using only the weak anomalous scattering from the endogenous S atoms. Moreover, a step-by-step investigation is presented of the particular problems of SAD phasing of SFX data, analysing data from a derivative with a strong anomalous signal as well as the weak signal from endogenous S atoms

Data from: Genetics and evidence for balancing selection of a sex-linked colour polymorphism in a songbird

Colour polymorphisms play a key role in sexual selection and speciation, yet the mechanisms that generate and maintain them are not fully understood. Here, we use genomic and transcriptomic tools to identify the precise genetic architecture and evolutionary history of a sex-linked colour polymorphism in the Gouldian finch Erythrura gouldiae that is also accompanied by remarkable differences in behaviour and physiology. We find that differences in colour are associated with an ~72-kbp region of the Z chromosome in a putative regulatory region for follistatin, an antagonist of the TGF-β superfamily genes. The region is highly differentiated between morphs, unlike the rest of the genome, yet we find no evidence that an inversion is involved in maintaining the distinct haplotypes. Coalescent simulations confirm that there is elevated nucleotide diversity and an excess of intermediate frequency alleles at this locus. We conclude that this pleiotropic colour polymorphism is most probably maintained by balancing selection

Kim et al 2019

Source data files used to produce Figures in Kim et al. 201