X-Ray and FELUltrafast Sources in Protein and Virus Imaging from Structure to Function

Our  collaboratation has recently demonstrated serial snapshot femtosecond diffraction (SFX) from membrane protein nanocrystals using the world's first hard X-ray laser, the LCLS at SLAC near Stanford. Diffraction patterns from a single virus were also obtained and phased. By recording patterns at 100 Hz from a stream of fully-hydrated submicron nanocrystals, it has since been found that near-atomic resolution data are obtained prior to vaporization of each nanocrystal, with little evidence of radiation damage, using  pulses consisting of 10¹³ photons in less than 40 fs. The first "new biology" from LCLS work, new features in the ezyme cathepsin density map, will be discussed. We outrun damage, since the pulse terminates before damage due to the photoelectron cascade commences. The dose is about 100 times the Henderson safe dose. This makes possible analysis of invisible nanocrystals from difficult-to-crystallize material, and suggests many experiments in snapshot time-resolved chemistry. Pump-probe experiments on Photosystem  I - ferredoxin using our liquid jet particle injector were attempted in June 2010, in order  to provide a stroboscopic molecular movie. Shape-transform effects on nanocrystal Bragg spots, due to the full coherence of the LCLS,  require new methods of data analysis, since each snapshot pattern shows only partial reflections. We show that a suitable sum of intensity around Bragg spots over nanocrystal size and orientation converges to the wanted structure factors. The intensity oscillations between Bragg reflections, summed over crsytal size, also offers a new iterative solution to the phase problem, which does not risk biasing the PDB. Z. Kam  pointed out that SAX patterns recorded from particles frozen in space or time gain two-dimensional fluctuations, providing more information than conventional one-dimensional SAXS patterns. A sum of the angular correlation functions of  these patterns converges to the correlation function for one particle, which may be inverted using new iterative phasing methods. We have demonstrated this experimentally for two-dimensional data. Application of  this approach to XFEL data will be discussed, since, unlike current single-particle XFEL experiments, the Kam  method ensures 100% "hit rate".  Since temperature fall along the liquid jet, this method is well suited to snap-shot chemistry experiments involving protein reactions with a substrate.