Dynamic catcher for stabilization of high-viscosity extrusion jets

A `catcher' based on a revolving cylindrical collector is described. The simple and inexpensive device reduces free-jet instabilities inherent to high-viscosity extrusion injection, facilitating delivery of microcrystals for serial diffraction X-ray crystallography

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

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin

International audienceBacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event uponlight absorption is isomerization of the retinal chromophore. Here we used time-resolvedcrystallography at an X-ray free-electron laser to follow the structural changes inmultiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry andultrafast spectroscopy were used to identify a sequential two-photon absorption process,leading to excitation of a tryptophan residueflanking the retinal chromophore, as afirstmanifestation of multiphoton effects. We resolve distinct stages in the structural dynamics ofthe all-transretinal in photoexcited bR to a highly twisted 13-cisconformation. Other activesite sub-picosecond rearrangements include correlated vibrational motions of the electro-nically excited retinal chromophore, the surrounding amino acids and water molecules as wellas their hydrogen bonding network. These results show that this extended photo-activenetwork forms an electronically and vibrationally coupled system in bR, and most likely in allretinal proteins

Shock Damage Analysis in Serial Femtosecond Crystallography Data Collected at MHz X-ray Free-Electron Lasers

International audienceSerial femtosecond crystallography (SFX) data were recorded at the European X-ray free-electron laser facility (EuXFEL) with protein microcrystals delivered via a microscopic liquid jet. An XFEL beam striking such a jet may launch supersonic shock waves up the jet, compromising the oncoming sample. To investigate this efficiently, we employed a novel XFEL pulse pattern to nominally expose the sample to between zero and four shock waves before being probed. Analyzing hit rate, indexing rate, and resolution for diffraction data recorded at MHz pulse rates, we found no evidence of damage. Notably, however, this conclusion could only be drawn after careful identification and assimilation of numerous interrelated experimental factors, which we describe in detail. Failure to do so would have led to an erroneous conclusion. Femtosecond photography of the sample-carrying jet revealed critically different jet behavior from that of all homogeneous liquid jets studied to date in this manner

Influence of pump laser fluence on ultrafast structural changes in myoglobin

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer 1 . However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore 2-14 . As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern 15 whether this experimental approach 16 allows valid inferences to be drawn vis-à-vis biologically relevant single-photon-induced reactions 17 . Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics 15 ) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments 16 such that biologically relevant insight emerges

Influence of pump laser fluence on ultrafast myoglobin structural dynamics

International audienceHigh-intensity femtosecond pulses from an X-ray free-electron laser enable pump–probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer 1,2 . However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore 3–17 . As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern 18,19 whether this experimental approach 20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions 18,19 . Here we describe ultrafast pump–probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe–CO bond distance (predicted by recent quantum wavepacket dynamics 21 ) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump–probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump–probe experiments 20 such that mechanistically relevant insight emerges

MHz data collection of a microcrystalline mixture of different jack bean proteins

Design Type(s)protocol testing objectiveMeasurement Type(s)protein structure dataTechnology Type(s)x ray crystallographyFactor Type(s)Sample Characteristic(s)Canavalia Machine-accessible metadata file describing the reported data (ISA-Tab format

Megahertz data collection from protein microcrystals at an X-ray free-electron laser

The European X-ray free-electron laser (EuXFEL) in Hamburg is the first megahertz (MHz) repetition rate XFEL. Here the authors use lysozyme crystals and microcrystals from jack bean proteins and demonstrate that damage-free high quality data can be collected at a MHz repetition rate

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan , respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo -grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control