Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams

Nass, Karol; Foucar, L.; Barends, Thomas R. M.; Hartmann, Elisabeth; Botha, Sabine; Shoeman, Robert L.; Doak, R. Bruce; Alonso‐Mori, R.; Aquila, Andrew; Bajt, Saša; Barty, Anton; Bean, Richard; Beyerlein, Kenneth R.; Bublitz, Maike; Drachmann, N.D.; Gregersen, J.L.; Jönsson, Olof; Kabsch, Wolfgang; Kassemeyer, Stephan; Koglin, Jason E.; Krumrey, Michael; Mattle, D.; Messerschmidt, Marc; Nissen, Poul; Reinhard, Linda; Sitsel, Oleg; Sokaras, Dimosthenis; Williams, Garth J.; Hau‐Riege, Stefan P.; Tı̂mneanu, Nicuşor; Caleman, Carl; Chapman, Henry N.; Boutet, Sébastien; Schlichting, Ilme

doi:10.1107/s1600577515002349

Cited by 117 publications

(121 citation statements)

References 66 publications

(69 reference statements)

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“…One of the applications for such capabilities is the understanding of molecular fragmentation induced by x rays, a process of fundamental interest and a relevant factor for the radiation damage induced during the structural imaging of biomolecules at XFELs [30][31][32]. Molecular ionization and fragmentation follow absorption of a single synchrotron x ray [33][34][35] or absorption of multiple x rays using XFEL radiation [36][37][38][39][40].…”

Section: Introductionmentioning

confidence: 99%

Ultrafast x-ray-induced nuclear dynamics in diatomic molecules using femtosecond x-ray-pump–x-ray-probe spectroscopy

et al. 2016

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The capability of generating two intense, femtosecond x-ray pulses with a controlled time delay opens the possibility of performing time-resolved experiments for x-ray-induced phenomena. We have applied this capability to study the photoinduced dynamics in diatomic molecules. In molecules composed of low-Z elements, K-shell ionization creates a core-hole state in which the main decay mode is an Auger process involving two electrons in the valence shell. After Auger decay, the nuclear wave packets of the transient two-valence-hole states continue evolving on the femtosecond time scale, leading either to separated atomic ions or long-lived quasibound states. By using an x-ray pump and an x-ray probe pulse tuned above the K-shell ionization threshold of the nitrogen molecule, we are able to observe ion dissociation in progress by measuring the time-dependent kinetic energy releases of different breakup channels. We simulated the measurements on N 2 with a molecular dynamics model that accounts for K-shell ionization, Auger decay, and the time evolution of the nuclear wave packets. In addition to explaining the time-dependent feature in the measured kinetic energy release distributions from the dissociative states, the simulation also reveals the contributions of quasibound states.

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Section: Introductionmentioning

confidence: 99%

Ultrafast x-ray-induced nuclear dynamics in diatomic molecules using femtosecond x-ray-pump–x-ray-probe spectroscopy

et al. 2016

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“…Given the high dose per crystal used in the SFX measurements, we looked for evidence of predicted local radiation damage (13,14) in the vicinity of radiation-sensitive disulfide bonds. Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Simulations (20,21) and experiments (12)(13)(14) suggest that it should be possible to outrun radiation damage with short enough pulses using much higher X-ray intensities. Calculations with pulse durations as short as 10 fs predict the possibility of high-resolution structure determination from nanocrystals at doses greater than 10 GGy, and 1-TGy doses should be tolerable with subfemtosecond pulses (22).…”

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confidence: 99%

“…Serial femtosecond crystallography (SFX) overcomes radiation damage by using ultrabright X-ray pulses that are shorter in duration than most damage processes (9)(10)(11)(12)(13)(14), thereby vastly increasing the tolerable dose and thus the amount of information gathered before the specimen is destroyed. Commonly, crystals in a liquid stream are injected into the X-ray beam, and each crystal diffracts for only a few femtoseconds before being destroyed (12,15).…”

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Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser

Gati

Oberthüer

Yefanov

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm 3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm 3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.XFEL | nanocrystals | structural biology | bioimaging | SFX I maging of biomolecules using radiation of short enough wavelength to resolve individual atoms is limited by radiation damage, which destroys the very structure being measured. Energy absorption is unavoidable because the ratio of elastic scattering to damaging photoabsorption is an inherent property of atoms. Absorbed dose is therefore proportional to the scattered intensity, and thus the measured signal, used to determine the structure. The maximum tolerable dose that the sample can withstand fundamentally limits atomic resolution structure determination (1). The tolerable dose, and therefore damage, is a function of spatial resolution, with fine detail being destroyed first (2, 3). Radiation damage is typically overcome by distributing the dose over many identical copies of the same molecule, either using crystals containing many aligned copies of the same molecule, or by aligning images of individual molecules in the case of single particle electron microscopy. At room temperature, free radical production after a dose as small as 150 kGy (gray = J _ kg −1 absorbed energy) causes rapid decay of biological samples (4). This decay can be reduced by cooling the sample to below ∼120 K....

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“…Heavy atoms are of course critical to phasing by anomalous scattering, and their oxidation state and detailed stereochemistry within the complex metal centers at the active site of, for example, photosystem II are essential to overall function. There are indications that the Fe atoms in the two chemically identical 4Fe -4S clusters in ferredoxin from Clostridium differ in their extent of radiation damage from XFEL illumination (Nass et al, 2015). The molecules CH 3 I and C 6 H 5 I studied by Rudenko et al (2017) are very small by biological standards but it seems entirely likely that their general result will hold for heavy atoms in biological macromolecules: atoms that are chemically identical may not be identical in their propensity for radiation damage!…”

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confidence: 99%

Small crystals, fast dynamics and noisy data are indeed beautiful

Moffat

2017

Int Union Crystallogr J

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Free electron lasers represent a rare example of disruptive innovation in technology (Bower & Christensen, 1995). Such an innovation requires us to identify the ways in which the new technology differs significantly from the old and to decide how best to deploy that technology. Most importantly, we must consider what hitherto inaccessible scientific problems can now be successfully attacked, and reconsider what older problems can be better attacked. Free electron lasers had been developed at much longer wavelengths in the electromagnetic spectrum and used effectively for some time. It was however not a foregone conclusion that the self-amplified spontaneous emission process that underpins lasing would continue to hold for a hard X-ray FEL (XFEL), and that the roughly 100 m long undulator necessary for saturation of this lasing process could be constructed with the required precision. Even if lasing were achieved, it was not at all clear -particularly to reviewers ten years ago of grant applications to NIH in XFELbased structural biology -that the prodigious peak brilliance of hard X-ray pulses could be harnessed to conduct any useful scattering or spectroscopic experiments before all atoms became fully ionized and the sample was destroyed.We know how that all turned out: the world's first hard X-ray laser, the Linac Coherent Light Source at the Stanford Linear Accelerator Center, speedily achieved lasing and was a roaring success. More to the point for X-ray experimentalists, a brief time window in the femtosecond range turned out to exist -as predicted by a few far-sighted scientistsduring which scattering or spectroscopy data could be collected before the sample was indeed destroyed in the ultimate destructive experiment: 'diffraction before destruction ' (Chapman et al., 2011). As one of the pioneers of XFEL science in structural biology and its associated technologies, John Spence now presents a magisterial review (Spence, 2017) that illustrates his command of a large swath of the recent literature and of course, of the experiments that underlie it. The field is expanding so rapidly that this may turn out to be the last review that realistically covers the entire field. That expansion also illustrates that the field is quite immature and to many of us, wildly exciting. Developments are being vigorously pursued in such areas as the source itself (e.g. seeding; control of the bandpass of the X-ray spectrum or of the X-ray pulse structure), the X-ray optics (e.g. split-anddelay schemes to manipulate the X-ray pulse structure delivered to the sample; chirping of the pulse in time or space), the design of experiments (e.g. fixed target or jet-based modes of sample delivery; sample manipulation in dynamic experiments by rapid mixing, an intense pump pulse by a visible or IR laser, a temperature jump or an electric field jump; the combination of X-ray scattering and spectroscopic experiments), the detector and perhaps above all, in modes of analysis of the data. All are dealt with in the review, some at length, oth...

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Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams

Cited by 117 publications

References 66 publications

Ultrafast x-ray-induced nuclear dynamics in diatomic molecules using femtosecond x-ray-pump–x-ray-probe spectroscopy

Ultrafast x-ray-induced nuclear dynamics in diatomic molecules using femtosecond x-ray-pump–x-ray-probe spectroscopy

Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser

Small crystals, fast dynamics and noisy data are indeed beautiful

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