3D diffractive imaging of nanoparticle ensembles using an X-ray laser

We report the 3D structure determination of gold nanoparticles (AuNPs) by X-ray single particle imaging (SPI). Around 10 million diffraction patterns from gold nanoparticles were measured in less than 100 hours of beam time, more than 100 times the amount of data in any single prior SPI experiment, using the new capabilities of the European X-ray free electron laser which allow measurements of 1500 frames per second. A classification and structural sorting method was developed to disentangle the heterogeneity of the particles and to obtain a resolution of better than 3 nm. With these new experimental and analytical developments, we have entered a new era for the SPI method and the path towards close-to-atomic resolution imaging of biomolecules is apparent

Mapping the continuous reciprocal space intensity distribution of X-ray serial crystallography

Serial crystallography using X-ray free-electron lasers enables the collection of tens of thousands of measurements from an equal number of individual crystals, each of which can be smaller than 1 mmin size. This manuscript describesan alternativeway of handling diffraction data recorded by serial femtosecond crystallography, by mapping the diffracted intensities into three-dimensional reciprocal space rather than integrating each image in two dimensions as in theclassical approach. We call this procedure ‘three-dimensional merging’. This procedure retains information about asymmetry in Bragg peaks and diffracted intensities between Bragg spots. This intensity distribution can be used toextract reflection intensities for structure determination and opens up novel avenues for post-refinement, while observed intensity between Bragg peaks and peak asymmetry are of potential use in novel direct phasing strategies

Phasing coherently illuminated nanocrystals bounded by partial unit cells

With the use of highly coherent femtosecond X-ray pulses from a free-electron laser, it is possible to record protein nanocrystal diffraction patterns with far more information than is present in conventional crystallographic diffractiondata. It has been suggested that diffraction phases may be retrieved from such data via iterative algorithms, without the use of a priori information and without restrictions on resolution. Here, we investigate the extension of thisapproach to nanocrystals with edge terminations that produce partial unit cells, and hence cannot be described by a common repeating unit cell. In this situation, the phase problemdescribed in previouswork must be reformulated.We demonstrate an approximate solution to this phase problem for crystals with random edge terminations

An isomorphous replacement method for efficient de novo phasing for serial femtosecond crystallography

SACLAのX線自由電子レーザーを用いた新規タンパク質立体構造決定に世界で初めて成功. 京都大学プレスリリース. 2015-09-14.Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) holds great potential for structure determination of challenging proteins that are not amenable to producing large well diffracting crystals. Efficient de novo phasing methods are highly demanding and as such most SFX structures have been determined by molecular replacement methods. Here we employed single isomorphous replacement with anomalous scattering (SIRAS) for phasing and demonstrate successful application to SFX de novo phasing. Only about 20,000 patterns in total were needed for SIRAS phasing while single wavelength anomalous dispersion (SAD) phasing was unsuccessful with more than 80,000 patterns of derivative crystals. We employed high energy X-rays from SACLA (12.6 keV) to take advantage of the large anomalous enhancement near the LIII absorption edge of Hg, which is one of the most widely used heavy atoms for phasing in conventional protein crystallography. Hard XFEL is of benefit for de novo phasing in the use of routinely used heavy atoms and high resolution data collection

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