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.

X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions1–4. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma1. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval2. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source5. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.

The Stardust spacecraft collected thousands of particles from comet 81P/Wild 2 and returned them to Earth for laboratory study. The preliminary examination of these samples shows that the nonvolatile portion of the comet is an unequilibrated assortment of materials that have both presolar and solar system origin. The comet contains an abundance of silicate grains that are much larger than predictions of interstellar grain models, and many of these are high-temperature minerals that appear to have formed in the inner regions of the solar nebula. Their presence in a comet proves that the formation of the solar system included mixing on the grandest scales.

T heory predicts 1-4 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 × 10 13 W cm −2 pulse, containing 10 12 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 oversampling 5-9 , 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 one 10 .X-ray free-electron lasers (FELs) are expected to permit diffractive imaging at high resolutions of nanometre-to micrometre-sized objects without the need for crystalline periodicity in the sample [1][2][3][4] . Structural studies within this size domain are particularly important in materials science, biology and medicine. Radiation-induced damage and sample movement prevent the accumulation of high-resolution scattering signals for such samples in conventional experiments 11,12 . Damage is caused by energy deposited into the sample by the very probes used for imaging, for example photons, electrons or neutrons. At X-ray frequencies, inner-shell processes dominate the ionization of the sample; photoemission is followed by Auger or fluorescence emission and shake excitations. The energies of the ejected photoelectrons, Auger electrons and shake electrons differ from each other, and these electrons are released at different times, but within about ten femtoseconds, following photoabsorption 1,13 . Thermalization of the ejected electrons through collisional electron cascades is completed within 10-100 fs (refs 14,15). Heat transport, diffusion and radical reactions take place over some picoseconds to milliseconds.The effect of X-ray-induced sample damage on the recorded image or diffraction pattern could be substantially reduced, if we could collect diffraction data faster than the relevant damage processes 1,16 . This approach requires very short and very bright X-ray pulses, such as those expected from a short-wavelength FEL. However, the large amount of energy deposited into the sample by a focused FEL pulse will ultimately turn the sample into a plasma. The question is when exactly would this happen. There are no experiments with X-rays in the relevant time and intensity nature physics VOL 2 DECEMBER 2006 www.nature.com/naturephysics

Organics found in comet 81P/Wild 2 samples show a heterogeneous and unequilibrated distribution in abundance and composition. Some organics are similar, but not identical, to those in interplanetary dust particles and carbonaceous meteorites. A class of aromatic-poor organic material is also present. The organics are rich in oxygen and nitrogen compared with meteoritic organics. Aromatic compounds are present, but the samples tend to be relatively poorer in aromatics than are meteorites and interplanetary dust particles. The presence of deuterium and nitrogen-15 excesses suggest that some organics have an interstellar/protostellar heritage. Although the variable extent of modification of these materials by impact capture is not yet fully constrained, a diverse suite of organic compounds is present and identifiable within the returned samples.

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.

We have carried out a comprehensive survey of the isotopic compositions (H, B, C, N, O, S) of a suite of interplanetary dust particles (IDPs), including both cluster and individual particles. Isotopic imaging with the NanoSIMS shows the presence of numerous discrete hotspots that are strongly enriched in 15 Based on their N isotopic compositions, the IDPs studied here can be divided into two groups. One group is characterized as being "isotopically primitive" and consists of those IDPs that have anomalous bulk N isotopic compositions. These particles typically also contain numerous 15 N-rich hotspots, occasional C isotopic anomalies, and abundant presolar silicate grains. In contrast, the other "isotopically normal" IDPs have normal bulk N isotopic compositions and, although some contain 15 N-rich hotspots, none exhibit C isotopic anomalies and none contain presolar silicate or oxide grains. Thus, isotopically interesting IDPs can be identified and selected on the basis of their N isotopic compositions for further study. However, isotopes. This may be due to different effects of secondary processing on the isotopic systems involved.3

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.