Experimental Phasing of MicroED Data Using Radiation Damage - PubMed (original) (raw)

Experimental Phasing of MicroED Data Using Radiation Damage

Michael W Martynowycz et al. Structure. 2020.

Abstract

We previously demonstrated that microcrystal electron diffraction (MicroED) can be used to determine atomic-resolution structures from vanishingly small three-dimensional crystals. Here, we present an example of an experimentally phased structure using only MicroED data. The structure of a seven-residue peptide is solved starting from differences to the diffraction intensities induced by structural changes due to radiation damage. The same wedge of reciprocal space was recorded twice by continuous-rotation MicroED from a set of 11 individual crystals. The data from the first pass were merged to make a "low-dose dataset." The data from the second pass were similarly merged to form a "damaged dataset." Differences between these two datasets were used to identify a single heavy-atom site from a Patterson difference map, and initial phases were generated. Finally, the structure was completed by iterative cycles of modeling and refinement.

Keywords: MicroED; cryoEM; heavy metal phasing; radiation damage.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1.. Differences between the Low-Dose and Damaged Datasets at 2.5-Å Resolution

(A) Patterson maps for the native (top), and isomorphous differences at scaling factors 0.966 (top middle), 0.85 (bottom middle), and 0.8 (bottom). Blue line indicates the plane of the zinc atom shared between plots. (B) Difference Fourier map at 2.5-Å resolution using a scaling factor of 0.85. Patterson maps are displayed at the z = 22 slice. Difference Fourier map is displayed at the y = 22 slice. All slices are displayed with a 3σ cutoff and have contour steps at the 0.1σ level. Peaks and their corresponding locations are indicated by arrows.

Figure 2.. Locating the Heavy-Atom Substructure and Building Out the Structure

(A) Fourier difference maps between the damaged and undamaged structure of GSNQNNF using the phases of 6CLI at 2.5-Å resolution contoured at 3σ level (top). Initial phases generated at 2.5-Å resolution (middle), and the map using the initial phases extended to 1.4-Å resolution (bottom). (B) Density maps at 1.0σ contour starting from the initial phases (top) with structures of intermediate building steps until the final structure was completed at step 34 (bottom). All density maps are 2Fo-Fc maps contoured at 1.0σ and carved at 2-Å around the atomic centers.

References

1. Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, and Adams PD (2012). Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr 68, 352–367. -PMC -PubMed
1. Brünger AT (1992). Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–475. -PubMed
1. Ceska TA, and Henderson R (1990). Analysis of high-resolution electron diffraction patterns from purple membrane labelled with heavy-atoms. J. Mol. Biol 213, 539–560. -PubMed
1. Clabbers MTB, van Genderen E, Wan W, Wiegers EL, Gruene T, and Abrahams JP (2017). Protein structure determination by electron diffraction using a single three-dimensional nanocrystal. Acta Crystallogr. D Struct. Biol 73, 738–748. -PMC -PubMed
1. Clabbers MTB, Gruene T, van Genderen E, and Abrahams JP (2019). Reducing dynamical electron scattering reveals hydrogen atoms. Acta Crystallogr. A Found. Adv 75 (Pt 1), 82–93. -PMC -PubMed

Experimental Phasing of MicroED Data Using Radiation Damage - PubMed (original) (raw)