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

Yamashita K, Pan D, Okuda T, Sugahara M, Kodan A, Yamaguchi T, Murai T, Gomi K, Kajiyama N, Mizohata E, Suzuki M, Nango E, Tono K, Joti Y, Kameshima T, Park J, Song C, Hatsui T, Yabashi M, Iwata S, Kato H, Ago H, Yamamoto M, Nakatsu T - Sci Rep (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: RIKEN SPring-8 Center, Sayo, 679-5148, Japan.

ABSTRACT
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.

No MeSH data available.


CCano and CCanoref as a function of pattern numbers used for Monte-Carlo integration.CCano was calculated using two data sets grouped randomly. CCanoref is defined as in the main text. The figure was prepared using R36 with ggplot2 package37.
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f6: CCano and CCanoref as a function of pattern numbers used for Monte-Carlo integration.CCano was calculated using two data sets grouped randomly. CCanoref is defined as in the main text. The figure was prepared using R36 with ggplot2 package37.

Mentions: SFX data analysis using CrystFEL relies on the Monte-Carlo integration of Bragg reflections that partially intercept the Ewald sphere2021. This statistical extrapolation process favors a greater number of indexed diffraction patterns for better accuracy in the measurements of reflection intensities. The correlation coefficient (CCcalc) between I and improved as more diffraction patterns were used (Supplementary Fig. 2). We compared the correlation in anomalous difference, CCano, of two randomly divided data sets by changing the number of diffraction patterns. This provides a measure for the quality of the anomalous signal with respect to the number of diffraction patterns. The analysis showed that the anomalous signals were very weak; CCano was −0.066 using 10,000 patterns and remained relatively low, at 0.038 even with ~85,000 patterns. This explains why SAD phasing was unsuccessful. For more accurate analysis of anomalous signals, we introduced a new correlation coefficient termed CCanoref, which measures the consistency between I(+) − I(−) and . Here is the structure factor calculated from the LRE Hg-bound model. The model was refined against the derivative data of 10,000 patterns with the theoretical anomalous scattering contributions of the Hg atoms incorporated into the refinement. The noted monotonic increase in the CCanoref values for the increased number of diffraction patterns confirms the presence and enhancement of the anomalous signals (Fig. 6). This is also confirmed by the increased peak height of the anomalous difference Patterson map (Fig. 7).


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

Yamashita K, Pan D, Okuda T, Sugahara M, Kodan A, Yamaguchi T, Murai T, Gomi K, Kajiyama N, Mizohata E, Suzuki M, Nango E, Tono K, Joti Y, Kameshima T, Park J, Song C, Hatsui T, Yabashi M, Iwata S, Kato H, Ago H, Yamamoto M, Nakatsu T - Sci Rep (2015)

CCano and CCanoref as a function of pattern numbers used for Monte-Carlo integration.CCano was calculated using two data sets grouped randomly. CCanoref is defined as in the main text. The figure was prepared using R36 with ggplot2 package37.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4566134&req=5

f6: CCano and CCanoref as a function of pattern numbers used for Monte-Carlo integration.CCano was calculated using two data sets grouped randomly. CCanoref is defined as in the main text. The figure was prepared using R36 with ggplot2 package37.
Mentions: SFX data analysis using CrystFEL relies on the Monte-Carlo integration of Bragg reflections that partially intercept the Ewald sphere2021. This statistical extrapolation process favors a greater number of indexed diffraction patterns for better accuracy in the measurements of reflection intensities. The correlation coefficient (CCcalc) between I and improved as more diffraction patterns were used (Supplementary Fig. 2). We compared the correlation in anomalous difference, CCano, of two randomly divided data sets by changing the number of diffraction patterns. This provides a measure for the quality of the anomalous signal with respect to the number of diffraction patterns. The analysis showed that the anomalous signals were very weak; CCano was −0.066 using 10,000 patterns and remained relatively low, at 0.038 even with ~85,000 patterns. This explains why SAD phasing was unsuccessful. For more accurate analysis of anomalous signals, we introduced a new correlation coefficient termed CCanoref, which measures the consistency between I(+) − I(−) and . Here is the structure factor calculated from the LRE Hg-bound model. The model was refined against the derivative data of 10,000 patterns with the theoretical anomalous scattering contributions of the Hg atoms incorporated into the refinement. The noted monotonic increase in the CCanoref values for the increased number of diffraction patterns confirms the presence and enhancement of the anomalous signals (Fig. 6). This is also confirmed by the increased peak height of the anomalous difference Patterson map (Fig. 7).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: RIKEN SPring-8 Center, Sayo, 679-5148, Japan.

ABSTRACT
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.

No MeSH data available.