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/lp/springer_journal/entering-an-era-of-dynamic-structural-biology-HHgoJNZ0RM
- Publisher
- Springer Journals
- Copyright
- Copyright © 2018 by Orville et al.
- Subject
- Life Sciences; Life Sciences, general
- eISSN
- 1741-7007
- DOI
- 10.1186/s12915-018-0533-4
- Publisher site
- See Article on Publisher Site
Abstract
(Table 1)—these beamlines are also pushing the develop- A recent paper in BMC Biology presents a general ment of serial data collection methods that exploit smaller method for mix-and-inject serial crystallography, to samples and room temperature. For example, the VMXi facilitate the visualization of enzyme intermediates via beamline at Diamond can deploy a double multilayer time-resolved serial femtosecond crystallography (tr-SFX). monochromator (DMM) to deliver > 10 photons/s at − 3 They apply their method to resolve in near atomic detail 13 keV with an energy band-pass (ΔE/E) of ~ 5 × 10 the cleavage and inactivation of the antibiotic ceftriaxone (pink beam). Such a narrow polychromatic source delivers by a β-lactamase enzyme from Mycobacterium more X-ray photons on the sample and yields more Bragg tuberculosis. Their work demonstrates the general reflections at the detector per exposure compared to trad- applicability of time-resolved crystallography, from which itional monochromatic diffraction. Pink beam diffraction dynamic structures, at atomic resolution, can be obtained. experiments are also not as complex as Laue methods that See research article: https://bmcbiol.biomedcentral.com/ exploit broader polychromatic sources. VMXi is designed articles/10.1186/s12915-018-0524-5. for room-temperature measurements where the 300 kGy dose limit will be reached within ~ 30–100 μsusing the unattenuated, focused beam. The dose limit is typically Commentary described by a general loss of diffraction quality of the We are currently experiencing parallel step-changes in crystal, whereas local changes in the macromolecule (such structural biology. On the one hand, X-ray crystallography as reduction of a metal centre) often experience and cryo-EM are essential tools for structural biologists, X-ray-induced photophysical perturbations with orders of wherein the raw data are almost always derived from sam- magnitude less dose. Moreover, specific alterations of a ples held at 100 K. In particular, the rapid advances in metal centre or chromophore often result in changes to cryo-EM are clearly having a large impact across the com- their spectroscopic signal(s) and also provide a means to munity [1]. But, life is dynamic and function is not compat- follow and characterize mechanistically relevant perturba- ible with the cryogenic conditions. Incidentally and on the tionstothe sample. These typesof beamlines also typically other hand, X-ray free electron lasers (XFELs) offer new op- field the newest and fastest detectors operating at 750 Hz portunities because their unparalleled intensity makes it or more. Therefore, synchrotron characteristics enable possible for even submicron size crystals to yield high qual- time-resolved MX studies with macromolecular catalytic ity structures. To this end, serial femtosecond crystallog- systems operating in the μs/ms and longer time regime [4]. raphy (SFX) methods that exploit slurries of microcrystals For observing shorter time scales, smaller crystals, and the XFEL fs pulses are most often conducted at ambi- and samples that are radiation sensitive, a number of ent temperature. Therefore, SFX methods are well suited to XFELs are available (Table 1). For instance, the LCLS 12 13 couple structural biology with functional dynamics [2]. and European XFEL deliver 10 –10 photons/50 fs 2 − 3 Advanced synchrotron beamlines that produce pulse, in a ≤ 5×5 μm size beam, with 5 to 1 × 10 13 14 high-flux (10 –10 photons/s), microfocus beamlines band-pass in SASE mode. The Cornell–SLAC Pixel (≤ 5×5 μm ) are either in operation now or under Array Detector (CSPAD) matches the 120 Hz at LCLS, and the Adaptive Gain Integrating Pixel Detector Correspondence: allen.orville@diamond.ac.uk (AGIPD) at the European XFEL can collect 3250 im- The research article was published in BMC Biology 2018: 10.1186/s12915-018- ages per second from the 27,000 Hz pulses. LCLS-II 0524-5. Diamond Light Source, Research Complex at Harwell, and University of and LCLS-II-HE plan to deliver MHz X-ray pulse Oxford, Oxfordshire OX11 0DE, UK © Orville et al. 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Orville BMC Biology (2018) 16:55 Page 2 of 4 Table 1 Examples of synchrotron and XFEL sources for serial crystallography methods Diamond light source XFELs (~ 50 fs pulse duration) Beamline I04 I24 VMXi Eu.XFEL LCLS-II SACLA SwissFEL PAL XFEL 11 12 14 a 12 12 11 11 11 Photons/time 5 × 10 3× 10 >10 ~10 ≥ 10 2×10 ~7 ×10 2× 10 −1 − 1 − 1 − 1 − 1 − 1 − 1 − 1 (s ) (s ) (s ) (pulse ) (pulse ) (pulse ) (pulse ) (pulse ) Beam size (μm ) ~ 10 × 5 ~5 ×5 ≤ 5× 5 < 1–10 × 10 < 1–3× 3 1.3 ×1.3 <3 ×3 <3 ×3 b b c 6 d Detector/pulse (Hz) 25–100 (750) 25–100 750 27,000 120–10 60 100 60 a 12 Using a double multilayer monochromator (double crystal monochromator DCM > 2 × 10 ph/s) Dectris Eiger 4 M or region of interest within Eiger 16 M 2700 pulses in a 600-μs train (4.5 MHz) at 10 Hz d 6 6 LCLS-II (~ 2020, soft X-rays at 10 , hard at 120 Hz) and LCLS-II-HE (~ 2022, soft and hard X-rays at 10 Hz) frequencies and new detectors are anticipated to lever- Intense XFEL pulses destroy crystalline biological sam- age these capabilities. Consequently, synchrotron ples [5]. Fortunately, the structural information is sources complement XFEL sources and together they encoded in the photons of the X-ray diffraction pattern provide a wide range of experimental conditions (Fig. 1). travelling at the speed of light, whereas the sample ex- For samples that do not contain radiation-sensitive cen- plosion happens at roughly the speed of sound. Serial tres and catalyse reaction cycles that are relatively slow, femtosecond crystallography therefore requires that a then synchrotrons provide the appropriate capabilities. new sample is in place for each X-ray pulse. This is often When reaction cycles include redox active chromo- achieved by using flow-focusing gas dynamic virtual phores and/or very reactive, short-lived intermediates, nozzles (ff-GDVN) that create small liquid jets. To clear such as Fe(IV)=O oxoferryl, then XFEL facilities are more the debris from the interaction region and replenish new appropriate. In either case, it is important that the sample sample, the jets must continually inject new sample into is at physiological temperature if functionally relevant the interaction region at ~ 10–30 m/s at the LCLS deliv- dynamics are to be included in the study. ering 120 Hz pulses. Much faster jets are needed to fully Fig. 1 A comparison of the time scales at which different X-ray sources can be applied, and the phenomena they might be used to observe. Biochemical processes span orders of magnitude in time, with faster process linked to electron transfer and photoisomerization, and slower events linked to conformational changes. Substrate diffusion into micron-sized crystals is faster than the average enzyme turnover time. The X-ray dose and time required to elicit a spectroscopic change and an X-ray diffraction pattern to high resolution at a modern synchrotron are illustrated with Deinococcus radiodurans phytochrome (Dr-BphP) and myoglobin (Mb) Orville BMC Biology (2018) 16:55 Page 3 of 4 exploit the MHz repetition rates of the European XFEL more than one space group and unit cell packing to in- and the LCLS-II. These considerations also place size crease the likelihood of success for a MISC experiments. constraints on the crystals in the sample and, conse- This also needs to be balanced against the particular mech- quently, slurries of microcrystals are ideal. Furthermore, anistic question(s) to be addressed, and the resolution of a critical step to producing samples for SFX methods is the electron density maps achievable from each crystal very often observed as a shower of microcrystals during form. The manuscript also shows that single turnover reac- an initial crystallization trial of most macromolecular tion schemes rapidly produce a high product concentration samples. Flow-focusing GDVN methods also provide op- that can subsequently inhibit enzyme reactions as they portunities to add substrates via the outer, flow-focusing move from transient to steady-state kinetic regimes. fluid, directly to slurries of microcrystals within the cen- This year, about 700,000 people worldwide will die from tral focused jet [6]. This strategy is similar to a continu- drug-resistant infections. Professor Dame Sally Davies, the ous flow transient kinetic scheme. Micron-size crystals chief medical officer in the UK, said recently that “without will equilibrate via diffusion with small molecule sub- action we risk infection related mortality returning to strates on the μs time scale, which is much faster than pre-antibiotic levels by the mid-21st century”. An inde- the 60-ms average turnover time of enzymes. If electro- pendent review on antimicrobial resistance, chaired by static interactions between the enzyme and the sub- macroeconomist Jim O’Neill, suggests that by 2050 mor- strate(s) are present, then one could anticipate even tality rates could reach 10 million people per year or about faster equilibration times. Therefore, time-resolved SFX 1 out of every 1000 people [10]. The status quo in struc- methods (tr-SFX) that produce high quality electron ture–function analysis in structural biology is to use many density maps are generalizable. An area of particular im- separate samples, from which different types of data are portance is the creation of new tools and strategies to collected under different conditions that may be far from manipulate microcrystal samples that may provide a physiological. To this end, structure-based drug discovery means to correlate enzyme kinetics, spectroscopy, and and fragment-based screening are major strategies to time-resolved structural biology. bring new drugs to market. And yet, most new drugs fail The paper by Olmos et al. [7]inthisissue is amongthe because they lack efficacy. These strategies typically rely first to demonstrate that ff-GDVN mixing jets and tr-SFX upon ground-state crystal structures determined at 100 K. methods can be used to observe transient intermediates of Therefore, an emerging alternative that de-risks the path enzymes engaged in catalysis. They use the phrase ‘mix-an- towards new therapeutics is to study the entire reaction d-inject serial crystallography’ (MISC) and demonstrate the cycle at physiological temperature. Indeed, with temporal, capability with BlaC, a beta-lactamase from Mycobacterium dynamic and functional data linked to atomic models, it is tuberculosis, catalysing the ring cleavage of ceftriaxone, a likely that new mechanistic insights will suggest novel so-called third-generation cephalosporin antibiotic. They strategies to mediate or inhibit function. For instance, one collected tr-SFX datasets at 30, 100, 500 and 2000 ms time- might target a transient state revealed by tr-SFX methods points after mixing with substrate at the Linac Coherent that is not sufficiently populated or observed by traditional Light Source at SLAC National Accelerator Laboratory in ligand soak and cryo-cool approaches. California, USA. The resulting electron density maps reveal New X-ray sources create exciting new opportunities features that are modelled as reaction intermediates that and as a result we are entering an era of dynamic struc- build up and go away with reaction time after mixing. Re- tural biology. This is as much a concept as a set of tools to finements of the atomic models fit to 2.15–2.75 Å reso- collect as much data as possible, from every sample and lution maps are used to estimate the relative concentrations X-ray pulse, and enables one to create atomic resolution of the intermediates at the timepoints and are fit to a kin- ‘movies’ of macromolecules engaged in catalysis. Thus, etic model. Although these results are supported by elec- time-resolved crystallography, a longstanding frontier tron density maps alone, the addition of complementary challenge for the field, is achievable with serial methods at spectroscopic and catalytic studies from the same sample(s) XFELs and at advanced synchrotron beamlines. will become more readily available and will provide import- ant verification of critical reactive intermediates in future Funding AMO is supported by Diamond Light Source and a Strategic Award from the studies on a wide range of systems [8]. Wellcome Trust and the Biotechnology and Biological Sciences Research One of the important results of the study is the clear Council (grant 102593). demonstration that the crystal packing significantly impacts reaction rates. It makes logical sense that a crystal form Authors’ contributions with a smaller solvent content and narrower intra-lattice The author read and approved the final manuscript. channels will not equilibrate with substrates soaked into the lattice as fast as those with larger solvent content and Competing interests larger channels [9]. Consequently, one should evaluate The author declares that he has no competing interests. Orville BMC Biology (2018) 16:55 Page 4 of 4 Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Nobelprize.org. The Nobel Prize in Chemistry 2017. 2017. https://www. nobelprize.org/nobel_prizes/chemistry/laureates/2017/.Accessed 7 May2018. 2. Johansson LC, Stauch B, Ishchenko A, Cherezov V. A bright future for serial femtosecond crystallography with XFELs. Trends Biochem Sci. 2017;42:749– 62. https://doi.org/10.1016/j.tibs.2017.06.007. 3. Grimes JM, Hall DR, Ashton AW, Evans G, Owen RL, Wagner A, et al. Where is crystallography going? Acta Crystallogr D Struct Biol. 2018;74:152–66. https://doi.org/10.1107/S2059798317016709. 4. Meents A, Wiedorn MO, Srajer V, Henning R, Sarrou I, Bergtholdt J, et al. Pink-beam serial crystallography. Nat Commun. 2017;8:1281. https://doi.org/10.1038/s41467-017-01417-3. 5. Stan CA, Milathianaki D, Laksmono H, Sierra RG, McQueen TA, Messerschmidt M, et al. Liquid explosions induced by X-ray laser pulses. Nat Phys. 2016;12:966–71. https://doi.org/10.1038/Nphys3779. 6. Calvey GD, Katz AM, Schaffer CB, Pollack L. Mixing injector enables time- resolved crystallography with high hit rate at X-ray free electron lasers. Struct Dyn. 2016;3:054301. https://doi.org/10.1063/1.4961971. 7. Olmos JL Jr, Pandey S, Martin-Garcia JM, Calvey G, Katz A, Knoska J, et al. Enzyme intermediates captured “on-the-fly” by mix-and-inject serial crystallography. BMC Biol. 2018 https://doi.org/10.1186/s12915-018-0524-5. 8. FullerFD, GulS,ChatterjeeR,BurgieES, Young ID, Lebrette H, et al. Drop-on- demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers. Nat Methods. 2017;14:443–9. https://doi.org/10.1038/nmeth.4195. 9. Geremia S, Campagnolo M, Demitri N, Johnson LN. Simulation of diffusion time of small molecules in protein crystals. Structure. 2006;14:393–400. https://doi.org/10.1016/j.str.2005.12.007. 10. O’Neill J. Tackling drug-resistant infections globally: final report and recommendations. 2016. https://amr-review.org/sites/default/files/160525_ Final%20paper_with%20cover.pdf. Accessed 7 May 2018.
Journal
BMC Biology – Springer Journals
Published: May 31, 2018