Sasa Bajt

Femtosecond x-ray protein nanocrystallography

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 recorded. 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 Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. 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 processes. 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.

Femtosecond diffractive imaging with a soft-X-ray free-electron laser

Theory predicts1,2,3,4 that, with an ultrashort and extremely bright coherent X-ray pulse, a single diffraction pattern may be recorded from a large macromolecule, a virus or a cell before the sample explodes and turns into a plasma. Here we report the first experimental demonstration of this principle using the FLASH soft-X-ray free-electron laser. An intense 25 fs, 4×1013 W cm−2 pulse, containing 1012 photons at 32 nm wavelength, produced a coherent diffraction pattern from a nanostructured non-periodic object, before destroying it at 60,000 K. A novel X-ray camera assured single-photon detection sensitivity by filtering out parasitic scattering and plasma radiation. The reconstructed image, obtained directly from the coherent pattern by phase retrieval through oversampling5,6,7,8,9, shows no measurable damage, and is reconstructed at the diffraction-limited resolution. A three-dimensional data set may be assembled from such images when copies of a reproducible sample are exposed to the beam one by one10.

Natively Inhibited Trypanosoma brucei Cathepsin B Structure Determined by Using an X-ray Laser

Diffraction Before Destruction A bottleneck in x-ray crystallography is the growth of well-ordered crystals large enough to obtain high-resolution diffraction data within an exposure that limits radiation damage. Serial femtosecond crystallography promises to overcome these constraints by using short intense pulses that out-run radiation damage. A stream of crystals is flowed across the free-electron beam and for each pulse, diffraction data is recorded from a single crystal before it is destroyed. Redecke et al. (p. 227, published online 29 November; see the Perspective by Helliwell) used this technique to determine the structure of an enzyme from Trypanosoma brucei, the parasite that causes sleeping sickness, from micron-sized crystals grown within insect cells. The structure shows how this enzyme, which is involved in degradation of host proteins, is natively inhibited prior to activation, which could help in the development of parasite-specific inhibitors. In vivo crystallization and serial femtosecond crystallography reveal the structure of a sleeping sickness parasite protease. [Also see Perspective by Helliwell] 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 microprobe analysis of iron oxidation states in silicates and oxides using X-ray absorption near edge structure (XANES)

Abstract Initial results are reported on a new microprobe technique for determining Fe 3+ ∑Fe ratios in Fe-bearing minerals. The technique is based on the energy shift of a pre-edge peak in X-ray absorption near-edge structure (XANES) spectra obtained using the synchrotron X-ray fluorescence microprobe. A linear relationship between pre-edge peak energy and iron oxidation state was observed for oxide and silicate standards. Reasonable iron oxidation state results were obtained for pallasitic olivine, acmite, altered magnetites and synthetic wustite. Direct measurements of Fe 3+ ∑Fe in coexisting phases is feasible using this technique.

Improved reflectance and stability of Mo-Si multilayers

Commercial EUV lithographic systems require multilayers with higher reflectance and better stability than those published to date. This work represents our effort to meet these specifications. Interface- engineered Mo-Si multilayers with 70% reflectance and 0.545-nm bandwidth at 13.5-nm wavelength and 71% reflectance with 0.49-nm bandwidth at 12.7-nm wavelength were developed. These results were achieved with 50 bilayers. These new multilayers consist of alternating Mo and Si layers separated by thin boron carbide layers. Depositing boron carbide on the interfaces leads to reduction in molybdenum silicide formation of the Mo-on-si interfaces. Bilayer contraction is reduced by 30%, implying that there is less intermixing of Mo and si to form silicide. As a result, the Mo-on-si interfaces are sharper in interface-engineered multilayers than in standard Mo-Si multilayers. The optimum boron carbide thicknesses have been determined and appear to be different for the Mo-on-Si and Si-on-Mo interfaces. The best results were obtained with 0.4-nm-thick boron carbide layers for the Mo-on-Si interfaces and 0.25-nm-thick boron carbide layers for the Si-on-Mo interfaces. The increase in reflectance is consistent with multilayers having sharper and smoother interfaces. A significant improvement in oxidation resistance of EUV multilayers has been achieved with ruthenium-terminated Mo-Si multilayers. The best capping-layer design consists of a Ru layer separated from the top Si layer by a boron carbide diffusion barrier. This design achieves high reflectance and the best oxidation resistance during EUV exposure in a water-vapor (oxidizing) environment. Electron- beam exposures of 4.5 h (in an effort to simulate EUV exposure perturbation of the top layers) in the presence of 5x 10-7-Torr water-vapor partial pressure show no measurable reflectance loss and no increase in the oxide thickness of Ru-terminated multilayers.

Time-resolved protein nanocrystallography using an X-ray free-electron laser

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.

Infrared Spectroscopy of Comet 81P/Wild 2 Samples Returned by Stardust

Infrared spectra of material captured from comet 81P/Wild 2 by the Stardust spacecraft reveal indigenous aliphatic hydrocarbons similar to those in interplanetary dust particles thought to be derived from comets, but with longer chain lengths than those observed in the diffuse interstellar medium. Similarly, the Stardust samples contain abundant amorphous silicates in addition to crystalline silicates such as olivine and pyroxene. The presence of crystalline silicates in Wild 2 is consistent with mixing of solar system and interstellar matter. No hydrous silicates or carbonate minerals were detected, which suggests a lack of aqueous processing of Wild 2 dust.

In vivo protein crystallization opens new routes in structural biology

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.

In situ Chemical Speciation of Uranium in Soils and Sediments by Micro X-ray Absorption Spectroscopy.

There has been substantial interest recently in chemical speciation and species transformations of uranium in contaminated soils, sediments, and nuclear wastes, both from the standpoint of predicting its mobility to and within subsurface environments and for developing effective strategies for remediating contaminated sites. We exploited the microanalytical capabilities of beam line X-26A at Brookhaven National Laboratory to collect XANES and SXRF spectra on localized (50-300-[mu]m) regions within a number of U-contaminated soils and sediments. This provided specific information on U oxidation states, qualitative information on U-bonding environments, and information on associated elemental distributions. 26 refs., 4 figs.

Lipidic phase membrane protein serial femtosecond crystallography.

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.