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      Femtosecond X-ray protein nanocrystallography.

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          Abstract

          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.

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          Refinement of macromolecular structures by the maximum-likelihood method.

          This paper reviews the mathematical basis of maximum likelihood. The likelihood function for macromolecular structures is extended to include prior phase information and experimental standard uncertainties. The assumption that different parts of a structure might have different errors is considered. A method for estimating sigma(A) using 'free' reflections is described and its effects analysed. The derived equations have been implemented in the program REFMAC. This has been tested on several proteins at different stages of refinement (bacterial alpha-amylase, cytochrome c', cross-linked insulin and oligopeptide binding protein). The results derived using the maximum-likelihood residual are consistently better than those obtained from least-squares refinement.
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            First lasing and operation of an ångstrom-wavelength free-electron laser

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              Potential for biomolecular imaging with femtosecond X-ray pulses.

              Sample damage by X-rays and other radiation limits the resolution of structural studies on non-repetitive and non-reproducible structures such as individual biomolecules or cells. Cooling can slow sample deterioration, but cannot eliminate damage-induced sample movement during the time needed for conventional measurements. Analyses of the dynamics of damage formation suggest that the conventional damage barrier (about 200 X-ray photons per A2 with X-rays of 12 keV energy or 1 A wavelength) may be extended at very high dose rates and very short exposure times. Here we have used computer simulations to investigate the structural information that can be recovered from the scattering of intense femtosecond X-ray pulses by single protein molecules and small assemblies. Estimations of radiation damage as a function of photon energy, pulse length, integrated pulse intensity and sample size show that experiments using very high X-ray dose rates and ultrashort exposures may provide useful structural information before radiation damage destroys the sample. We predict that such ultrashort, high-intensity X-ray pulses from free-electron lasers that are currently under development, in combination with container-free sample handling methods based on spraying techniques, will provide a new approach to structural determinations with X-rays.
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                Author and article information

                Affiliations
                [1 ] Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany. henry.chapman@desy.de
                Journal
                Nature
                Nature
                1476-4687
                0028-0836
                Feb 3 2011
                : 470
                : 7332
                nature09750 NIHMS394803
                10.1038/nature09750
                3429598
                21293373

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