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An environmentally safe means of mosquito control is the application of Bacillus thuringiensis israelensis, which produces a cocktail of four naturally crystalline proteins exclusively toxic to mosquito. Here the authors report the atomic-resolution structures of Bti Cry11Aa and related Btj Cry11Ba solved de novo through Serial Femtosecond Crystallography on naturally-occurring nanocrystals.

Femtosecond crystallography with an X-ray free-electron laser is used to analyse micrometre-sized protein crystals, generating a high-resolution structure of the protein without previous knowledge of what it looks like.

Researchers describe a mechanism capable of compressing fast and intense X-ray pulses through the rapid loss of crystalline periodicity. It is hoped that this concept, combined with X-ray free-electron laser technology, will allow scientists to obtain structural information at atomic resolutions.

Lipidic sponge phase crystallization yields membrane protein microcrystals that can be injected into an X-ray free electron laser beam, yielding diffraction patterns that can be processed to recover the crystal structure.

The local X-ray-induced dynamics that occur in protein crystals during serial femtosecond crystallography (SFX) measurements at XFELs are not well understood. Here the authors performed a time-resolved X-ray pump X-ray probe SFX experiment, and they observe distinct structural changes in the disulfide bridges and peptide backbone of proteins; complementing theoretical approaches allow them to further characterize the details of the X-ray induced ionization and local structural dynamics.

Serial femtosecond crystallography is an X-ray free-electron-laser-based method that uses X-ray bursts to determine protein structures. Here the authors present the structure of a photosynthetic reaction centre, an integral membrane protein, achieved with no sign of X-ray-induced radiation damage.

Ultrafast time-resolved serial femtosecond crystallography is used to investigate a photodissociation reaction in a protein, revealing the strong impact of the pump laser fluence on the structural changes  and the reaction mechanism.

Serial femtosecond X-ray crystallography permits the use of very small protein crystals; however, a continuous flow of sample is required. Weierstall et al. design and demonstrate an injector system that can supply microcrystals in the lipidic cubic phase, dramatically reducing the quantities of protein required.

X-ray fee-electron lasers (XFELs) enable time-resolved crystallography experiments and the structure determination of proteins with little or no radiation damage. However currently it is unknown whether the designated 4.5 MHz maximum pulse rate for the European XFEL could lead to sample damage caused by shock waves from preceding pulses. Here, the authors address this question by performing a X-ray pump X-ray probe experiment on haemoglobin microcrystals at the Stanford XFEL facility that mimics the 4.5 MHz data collection mode and observe structural changes and a drop in diffraction data quality of the crystals.

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.

rsEGFP2 is a reversibly photoswitchable fluorescent protein used in super-resolution light microscopy. Here the authors present the structure of an rsEGFP2 ground-state intermediate after excited state-decay that was obtained by nanosecond time-resolved serial femtosecond crystallography at an X-ray free electron laser, and time-resolved absorption spectroscopy measurements complement their structural analysis.

X-ray free-electron lasers produce bright femtosecond X-ray pulses. Here, the authors use a two-colour X-ray free-electron laser beam for simultaneous two-wavelength data collection and show that protein structures can be determined with multiple wavelength anomalous dispersion phasing, which is important for difficult-to-phase projects.

Bacteriorhodopsin (bR) is a light-driven proton pump. Here the authors combine time-resolved crystallography at a free-electron laser, ultrafast spectroscopy and quantum chemistry to study the structural changes following multiphoton photoexcitation of bR and find that they occur within 300 fs not only in the light-absorbing chromophore but also in the surrounding protein.

Providing detailed structural descriptions of the ultrafast photochemical events that occur in light-sensitive proteins is key to their understanding. Now, excited-state structures in the reversibly switchable fluorescent protein rsEGFP2 have been solved by time-resolved crystallography using an X-ray laser. These structures enabled the design of a mutant with improved photoswitching quantum yields.

The new European X-Ray Free-Electron Laser (EuXFEL) is the first XFEL that generates X-ray pulses with a megahertz inter-pulse spacing. Here the authors demonstrate that high-quality and damage-free protein structures can be obtained with the currently available 1.1 MHz repetition rate pulses using lysozyme as a test case and furthermore present a β-lactamase structure.

Using resonance Raman spectroscopy and serial femtosecond X-ray crystallography, the authors show the heme a₃ iron and Cu_B in the resting oxidized form of Cytochrome c Oxidase are coordinated by a hydroxide ion and a water molecule, respectively.

Theoretical and experimental guidelines to determine laser intensity for single-photon excitation in microcrystals for ultrafast pump–probe experiments by serial femtosecond crystallography.

The start-up of the new femtosecond hard X-ray laser facility in Stanford, the Linac Coherent Light Source, has brought high expectations for a new era for biological imaging. The intense, ultrashort X-ray pulses allow diffraction imaging of small structures before radiation damage occurs. This new capability is tested for the problem of structure determination from nanocrystals of macromolecules that cannot be grown in large crystals. Over three million diffraction patterns were collected from a stream of nanocrystals of the membrane protein complex photosystem I, which allowed the assembly of a three-dimensional data set for this protein, and proves the concept of this imaging technique.

The start-up of the new femtosecond hard X-ray laser facility in Stanford, the Linac Coherent Light Source, has brought high expectations for a new era for biological imaging. The intense, ultrashort X-ray pulses allow diffraction imaging of small structures before radiation damage occurs. This new capability is tested for the problem of imaging a non-crystalline biological sample. Images of mimivirus are obtained, the largest known virus with a total diameter of about 0.75 micrometres, by injecting a beam of cooled mimivirus particles into the X-ray beam. The measurements indicate no damage during imaging and prove the concept of this imaging technique.

Expression of a protein in Sf9 insect cells at high concentration triggers formation of in vivo crystals that can be analyzed by serial femtosecond X-ray crystallography.

Femtosecond X-ray pulses were used to obtain diffraction data on photosystem II, revealing conformational changes as the complex transitions from the dark S₁ state to the double-pumped S₃ state; the time-resolved serial femtosecond crystallography technique enables structural determination of protein conformations that are highly prone to traditional radiation damage.

A 'protein quake' is directly monitored on the picosecond timescale using the method of time-resolved wide-angle X-ray scattering at an X-ray free-electron laser.