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Towards phasing using high X-ray intensity.

Galli L, Son SK, Barends TR, White TA, Barty A, Botha S, Boutet S, Caleman C, Doak RB, Nanao MH, Nass K, Shoeman RL, Timneanu N, Santra R, Schlichting I, Chapman HN - IUCrJ (2015)

Bottom Line: X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography.The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential 'bleaching' of heavy atoms.A pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY , Notkestrasse 85, Hamburg, 22607, Germany ; Department of Physics, University of Hamburg , Luruper Chaussee 149, Hamburg, 22761, Germany.

ABSTRACT
X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential 'bleaching' of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. A pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed.

No MeSH data available.


Related in: MedlinePlus

(a) Scatter plot of the average intensity of found peaks against the pulse energy, for the high-fluence data set. Each point corresponds to a single indexed diffraction pattern. The colours refer to the number of Bragg peaks found in the pattern (also shown in the upper-right plot, as a function of the maximum resolution found in the corresponding diffraction pattern). The black curves are the projected histograms of the values of the corresponding axis. (b) Discrete density plot of the number of found peaks versus the highest resolution found. Each hexagonal cell is coloured corresponding to the frequency of patterns in that region.
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fig3: (a) Scatter plot of the average intensity of found peaks against the pulse energy, for the high-fluence data set. Each point corresponds to a single indexed diffraction pattern. The colours refer to the number of Bragg peaks found in the pattern (also shown in the upper-right plot, as a function of the maximum resolution found in the corresponding diffraction pattern). The black curves are the projected histograms of the values of the corresponding axis. (b) Discrete density plot of the number of found peaks versus the highest resolution found. Each hexagonal cell is coloured corresponding to the frequency of patterns in that region.

Mentions: Due to the stochastic nature of the FEL operation, and the uncertain position, size and shape of the focus, the nominal ‘high-fluence’ data set is aggregated from a mixture of different fluences and therefore a mixture of doses. A similar but less dramatic result applies to the low-fluence data set, since the fluence is not high enough to cause a significant change of the scattering factors. In order to optimize the difference signal in the single-wavelength HIP method, the difference between the X-ray fluences must be the highest possible. To achieve this, we sorted the indexed diffraction snapshots to select only the patterns with the highest fluence. The narrow size distribution of the lysozyme microcrystals means that the observed diffracted intensity should be proportional to the fluence impinging on the crystal (see Lomb et al., 2011 ▸) except for the consideration of the beam’s spatial profile as discussed above. We used the number and average integrated intensity of peaks detected in the patterns, combined with readings from a pulse intensity monitor located upstream of the focusing mirror, to find the snapshots corresponding to the highest dose. These values are represented as a scatter plot in Fig. 3 ▸(a), showing a correlation between the number and the average peak intensity on the one hand, and the beam energy on the other. In particular, bright diffraction patterns are mostly found for high-intensity X-ray pulses, and often present a large number of Bragg spots. Furthermore, a high number of Bragg peaks also favours the highest-resolution patterns, as shown in Fig. 3 ▸(b), probably selecting also the best diffracting crystals. These brighter diffraction patterns were selected from the HF set as described in the supporting information, from which a new data set of 121 917 patterns labelled ‘HF_best’ was created. This set still presented a satisfactory data quality (see the third column of Table 1 ▸ and the comparison reported in Table 2 ▸). The previous analysis was repeated comparing the new HF_best to the full LF set, showing a higher ionization degree of the Gd atoms, corresponding to 12e−, consistent with the difference maps that also showed peaks at slightly higher sigma levels (9.2 and 6.3σ).


Towards phasing using high X-ray intensity.

Galli L, Son SK, Barends TR, White TA, Barty A, Botha S, Boutet S, Caleman C, Doak RB, Nanao MH, Nass K, Shoeman RL, Timneanu N, Santra R, Schlichting I, Chapman HN - IUCrJ (2015)

(a) Scatter plot of the average intensity of found peaks against the pulse energy, for the high-fluence data set. Each point corresponds to a single indexed diffraction pattern. The colours refer to the number of Bragg peaks found in the pattern (also shown in the upper-right plot, as a function of the maximum resolution found in the corresponding diffraction pattern). The black curves are the projected histograms of the values of the corresponding axis. (b) Discrete density plot of the number of found peaks versus the highest resolution found. Each hexagonal cell is coloured corresponding to the frequency of patterns in that region.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4645107&req=5

fig3: (a) Scatter plot of the average intensity of found peaks against the pulse energy, for the high-fluence data set. Each point corresponds to a single indexed diffraction pattern. The colours refer to the number of Bragg peaks found in the pattern (also shown in the upper-right plot, as a function of the maximum resolution found in the corresponding diffraction pattern). The black curves are the projected histograms of the values of the corresponding axis. (b) Discrete density plot of the number of found peaks versus the highest resolution found. Each hexagonal cell is coloured corresponding to the frequency of patterns in that region.
Mentions: Due to the stochastic nature of the FEL operation, and the uncertain position, size and shape of the focus, the nominal ‘high-fluence’ data set is aggregated from a mixture of different fluences and therefore a mixture of doses. A similar but less dramatic result applies to the low-fluence data set, since the fluence is not high enough to cause a significant change of the scattering factors. In order to optimize the difference signal in the single-wavelength HIP method, the difference between the X-ray fluences must be the highest possible. To achieve this, we sorted the indexed diffraction snapshots to select only the patterns with the highest fluence. The narrow size distribution of the lysozyme microcrystals means that the observed diffracted intensity should be proportional to the fluence impinging on the crystal (see Lomb et al., 2011 ▸) except for the consideration of the beam’s spatial profile as discussed above. We used the number and average integrated intensity of peaks detected in the patterns, combined with readings from a pulse intensity monitor located upstream of the focusing mirror, to find the snapshots corresponding to the highest dose. These values are represented as a scatter plot in Fig. 3 ▸(a), showing a correlation between the number and the average peak intensity on the one hand, and the beam energy on the other. In particular, bright diffraction patterns are mostly found for high-intensity X-ray pulses, and often present a large number of Bragg spots. Furthermore, a high number of Bragg peaks also favours the highest-resolution patterns, as shown in Fig. 3 ▸(b), probably selecting also the best diffracting crystals. These brighter diffraction patterns were selected from the HF set as described in the supporting information, from which a new data set of 121 917 patterns labelled ‘HF_best’ was created. This set still presented a satisfactory data quality (see the third column of Table 1 ▸ and the comparison reported in Table 2 ▸). The previous analysis was repeated comparing the new HF_best to the full LF set, showing a higher ionization degree of the Gd atoms, corresponding to 12e−, consistent with the difference maps that also showed peaks at slightly higher sigma levels (9.2 and 6.3σ).

Bottom Line: X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography.The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential 'bleaching' of heavy atoms.A pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY , Notkestrasse 85, Hamburg, 22607, Germany ; Department of Physics, University of Hamburg , Luruper Chaussee 149, Hamburg, 22761, Germany.

ABSTRACT
X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential 'bleaching' of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. A pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed.

No MeSH data available.


Related in: MedlinePlus