Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.
The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the “diffraction-before-destruction” approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.
X-ray free-electron lasers deliver intense femtosecond pulses that promise to yield high resolution diffraction data of nanocrystals before the destruction of the sample by radiation damage. Diffraction intensities of lysozyme nanocrystals collected at the Linac Coherent Light Source using 2 keV photons were used for structure determination by molecular replacement and analyzed for radiation damage as a function of pulse length and fluence. Signatures of radiation damage are observed for pulses as short as 70 fs. Parametric scaling used in conventional crystallography does not account for the observed effects.
X-ray free-electron lasers have enabled new approaches to the structural determination of protein crystals that are too small or radiation-sensitive for conventional analysis1. For sufficiently short pulses, diffraction is collected before significant changes occur to the sample, and it has been predicted that pulses as short as 10 fs may be required to acquire atomic-resolution structural information1–4. Here, we describe a mechanism unique to ultrafast, ultra-intense X-ray experiments that allows structural information to be collected from crystalline samples using high radiation doses without the requirement for the pulse to terminate before the onset of sample damage. Instead, the diffracted X-rays are gated by a rapid loss of crystalline periodicity, producing apparent pulse lengths significantly shorter than the duration of the incident pulse. The shortest apparent pulse lengths occur at the highest resolution, and our measurements indicate that current X-ray free-electron laser technology5 should enable structural determination from submicrometre protein crystals with atomic resolution.
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
(170.7160) Ultrafast technology; (170.7440) X-ray imaging; (140.3450) Laser-induced chemistry; (140.7090) Ultrafast lasers; (170.0170) Medical optics and biotechnology
Below-ground (BG) symbionts of plants can have substantial influence on plant growth and nutrition. Recent work demonstrates that mycorrhizal fungi can affect plant resistance to herbivory and the performance of above- (AG) and BG herbivores. Although these examples emerge from diverse systems, it is unclear if plant species that express similar defensive traits respond similarly to fungal colonization, but comparative work may inform this question. To examine the effects of arbuscular mycorrhizal fungi (AMF) on the expression of chemical resistance, we inoculated 8 species of Asclepias (milkweed)—which all produce toxic cardenolides—with a community of AMF. We quantified plant biomass, foliar and root cardenolide concentration and composition, and assessed evidence for a growth-defense tradeoff in the presence and absence of AMF. As expected, total foliar and root cardenolide concentration varied among milkweed species. Importantly, the effect of mycorrhizal fungi on total foliar cardenolide concentration also varied among milkweed species, with foliar cardenolides increasing or decreasing, depending on the plant species. We detected a phylogenetic signal to this variation; AMF fungi reduced foliar cardenolide concentrations to a greater extent in the clade including A. curassavica than in the clade including A. syriaca. Moreover, AMF inoculation shifted the composition of cardenolides in AG and BG plant tissues in a species-specific fashion. Mycorrhizal inoculation changed the relative distribution of cardenolides between root and shoot tissue in a species-specific fashion, but did not affect cardenolide diversity or polarity. Finally, a tradeoff between plant growth and defense in non-mycorrhizal plants was mitigated completely by AMF inoculation. Overall, we conclude that the effects of AMF inoculation on the expression of chemical resistance can vary among congeneric plant species, and ameliorate tradeoffs between growth and defense.
plant–herbivore interactions; mycorrhizal fungi; plant defense; above-below-ground interactions; growth-defense tradeoff; root defense; phylogenetic signal
Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet brought fully-hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses (~ 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. During the initial experiments at the AMO beamline using 6.9-Å wavelength, Bragg peaks were recorded to 8.5-Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage [Chapman H.N., et al. Femtosecond X-ray protein nanocrystallography, Nature 470 (2011) 73–81]. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.
membrane proteins; structure determination; femtosecond nanocrystallography; protein nanocrystals; X-ray crystallography; XFEL
X-ray free electron laser (X-feL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-feL beam using a sponge phase micro-jet.
X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded1-3. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction ‘snapshots’ are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source4. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes5. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (~200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes6. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo–grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 μs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
A complete set of structure factors has been extracted from hundreds of thousands of femtosecond X-ray diffraction patterns from randomly oriented Photosystem I membrane protein nanocrystals, using the Monte Carlo method of intensity integration. The data, collected at the Linac Coherent Light Source, are compared with conventional single-crystal data collected at a synchrotron source, and the quality of each data set was found to be similar.
A complete set of structure factors has been extracted from hundreds of thousands of femtosecond single-shot X-ray microdiffraction patterns taken from randomly oriented nanocrystals. The method of Monte Carlo integration over crystallite size and orientation was applied to experimental data from Photosystem I nanocrystals. This arrives at structure factors from many partial reflections without prior knowledge of the particle-size distribution. The data were collected at the Linac Coherent Light Source (the first hard-X-ray laser user facility), to which was fitted a hydrated protein nanocrystal injector jet, according to the method of serial crystallography. The data are single ‘still’ diffraction snapshots, each from a different nanocrystal with sizes ranging between 100 nm and 2 µm, so the angular width of Bragg peaks was dominated by crystal-size effects. These results were compared with single-crystal data recorded from large crystals of Photosystem I at the Advanced Light Source and the quality of the data was found to be similar. The implications for improving the efficiency of data collection by allowing the use of very small crystals, for radiation-damage reduction and for time-resolved diffraction studies at room temperature are discussed.
nanocrystals; femtosecond diffraction; free-electron lasers; Monte Carlo methods; protein microdiffraction
The cytoplasmic N–terminal domain of the human ether–a–go–go related gene (hERG) K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s) by which the N–terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N–terminal 135 residues of hERG contains a previously described Per–Arnt–Sim (PAS) domain (residues 26–135) as well as an amphipathic α–helix (residues 13–23) and an initial unstructured segment (residues 2–9). Deletion of residues 2–25, only the unstructured segment (residues 2–9) or replacement of the α–helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α–helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N–terminal α–helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.