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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

Phased difference (Fo − Fc) Fourier map, superposed to the lysozyme model deprived of the two Gd ions. Data to 2.1 Å, contoured at 4σ.
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fig1: Phased difference (Fo − Fc) Fourier map, superposed to the lysozyme model deprived of the two Gd ions. Data to 2.1 Å, contoured at 4σ.

Mentions: The resolution-dependent attenuation of the Si attenuator was corrected in the HF data set after the Monte Carlo integration process by dividing each reflection’s intensity by the calculated transmission factor at the corresponding scattering angle. Structure factors were calculated for the HF and LF data sets using CCP4 Truncate (French & Wilson, 1978 ▸) with default options. Cross scaling was performed with CCP4 Scaleit, treating the high-fluence data as native and the low-fluence as derivative, since the more heavily ionized Gd atoms, with fewer electrons, can be considered as lighter elements. To visualize the difference in the signal of the Gd atoms, an Fo − Fo difference density map was calculated using the lysozyme phases obtained by molecular replacement, performed with Phaser (McCoy et al., 2007 ▸). As search model, the structure of Gd-derivatized lysozyme (Protein Data Bank code 1h87, Girard et al., 2002 ▸) was used after the removal of the Gd ions. The map, displayed in Fig. 1 ▸, shows two high peaks at the Gd locations. One peak is higher than the other (9.0σ versus 6.2σ), probably due to the higher occupancy of the site (Girard et al., 2002 ▸). In order to estimate the relative number of electrons making up the difference between the two data sets at the Gd positions, two separate molecular-replacement runs were performed, using a search model from which the two Gd ions and part of a tryptophan (Trp) residue (48 electrons in total) had been removed (see the supporting information for details). No significant change in the B factors (global and local around the omitted regions) was observed in the two separately refined structures. This finding is important for a quantitative comparison of the electron densities of the omitted parts. Fo − Fc maps were calculated around the two missing regions, and these positive difference electron densities were volume integrated. The ratio between the integrated densities around the Gd and the Trp, multiplied by the number of missing electrons at the Trp location, gives an estimate of the effective scattering strength of the two Gd ions. Considering the average occupancy of the two sites, we found that the difference between the two data sets was around 8.8e− per Gd. By repeating the same procedure with other Trp present in the protein, an estimation was made of the error associated with the number of electrons, which is around 20%.


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)

Phased difference (Fo − Fc) Fourier map, superposed to the lysozyme model deprived of the two Gd ions. Data to 2.1 Å, contoured at 4σ.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Phased difference (Fo − Fc) Fourier map, superposed to the lysozyme model deprived of the two Gd ions. Data to 2.1 Å, contoured at 4σ.
Mentions: The resolution-dependent attenuation of the Si attenuator was corrected in the HF data set after the Monte Carlo integration process by dividing each reflection’s intensity by the calculated transmission factor at the corresponding scattering angle. Structure factors were calculated for the HF and LF data sets using CCP4 Truncate (French & Wilson, 1978 ▸) with default options. Cross scaling was performed with CCP4 Scaleit, treating the high-fluence data as native and the low-fluence as derivative, since the more heavily ionized Gd atoms, with fewer electrons, can be considered as lighter elements. To visualize the difference in the signal of the Gd atoms, an Fo − Fo difference density map was calculated using the lysozyme phases obtained by molecular replacement, performed with Phaser (McCoy et al., 2007 ▸). As search model, the structure of Gd-derivatized lysozyme (Protein Data Bank code 1h87, Girard et al., 2002 ▸) was used after the removal of the Gd ions. The map, displayed in Fig. 1 ▸, shows two high peaks at the Gd locations. One peak is higher than the other (9.0σ versus 6.2σ), probably due to the higher occupancy of the site (Girard et al., 2002 ▸). In order to estimate the relative number of electrons making up the difference between the two data sets at the Gd positions, two separate molecular-replacement runs were performed, using a search model from which the two Gd ions and part of a tryptophan (Trp) residue (48 electrons in total) had been removed (see the supporting information for details). No significant change in the B factors (global and local around the omitted regions) was observed in the two separately refined structures. This finding is important for a quantitative comparison of the electron densities of the omitted parts. Fo − Fc maps were calculated around the two missing regions, and these positive difference electron densities were volume integrated. The ratio between the integrated densities around the Gd and the Trp, multiplied by the number of missing electrons at the Trp location, gives an estimate of the effective scattering strength of the two Gd ions. Considering the average occupancy of the two sites, we found that the difference between the two data sets was around 8.8e− per Gd. By repeating the same procedure with other Trp present in the protein, an estimation was made of the error associated with the number of electrons, which is around 20%.

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