Research Article: Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER

Date Published: August 01, 2020

Publisher: International Union of Crystallography

Author(s): Logan S. Richards, Claudia Millán, Jennifer Miao, Michael W. Martynowycz, Michael R. Sawaya, Tamir Gonen, Rafael J. Borges, Isabel Usón, Jose A. Rodriguez.

http://doi.org/10.1107/S2059798320008049

Abstract

A 1.6 Å resolution MicroED data set of proteinase K is phased using fragments derived from distantly related sequence homologues. ARCIMBOLDO_SHREDDER expands the phasing options for MicroED applications, overcoming the need for complete and highly accurate search models.

Partial Text

Crystallography has remained an indispensable method for structure determination since its initial demonstration over a century ago (Bragg & Bragg, 1913 ▸). Beyond X-ray diffraction, neutron and electron diffraction have contributed important advances to the crystallographic determination of macromolecular structures (Glaeser, 1999 ▸; Shi et al., 2013 ▸; Gemmi et al., 2019 ▸). Recently, an electron crystallography method called microcrystal electron diffraction (MicroED) has been developed to obtain high-resolution structures from frozen-hydrated three-dimensional macromolecular crystals (Supplementary Fig. S1; Nannenga, Shi, Hattne et al., 2014 ▸). In MicroED, crystals of a few hundred nanometres in thickness are continuously rotated in an electron beam while diffraction is measured from a region of the specimen defined by the selected area aperture; the latter is positioned at the conjugate image plane of the objective lens. The recorded diffraction is reduced using conventional X-ray crystallography software to yield data that are suitable for structure determination. Phasing of MicroED data for biomolecules has been achieved by three approaches: by molecular replacement (Shi et al., 2013 ▸), by direct methods (Sawaya et al., 2016 ▸) or by using radiation damage (Martynowycz et al., 2020 ▸). Refinement proceeds through programs such as REFMAC (Kovalevskiy et al., 2018 ▸), phenix.refine (Afonine et al., 2012 ▸) or SHELXL (Sheldrick, 2015b ▸) using electron scattering factors.

As the field of MicroED continues to expand, a growing number of novel structures may present phasing hurdles. Given that experimental phasing remains a challenge in MicroED, it is important to explore other ways to overcome the phase problem beyond direct methods and molecular replacement. To date, more than a dozen ab initio structures determined by direct methods from MicroED data have been deposited in the PDB, in comparison to several dozen structures determined by conventional molecular replacement with resolutions between 1.2 and 3 Å (Rodriguez & Gonen, 2016 ▸). Of the set determined by molecular replacement, approximately 13 are in some way novel, although many of these rely on highly similar search models determined by X-ray diffraction. The relatively low number of completely novel structures is due in part to the challenges associated with the experimental phasing of MicroED data. Given the smaller difference in scattering between heavy and light atoms in electron diffraction compared with X-ray diffraction, experimental phasing by isomorphous replacement remains un­demonstrated and, at least for 2D crystals, might be intractable (Ceska & Henderson, 1990 ▸).

The following references are cited in the supporting information for this article: Arndt & Wonacott (1977 ▸), Nannenga & Gonen (2016 ▸).

 

Source:

http://doi.org/10.1107/S2059798320008049

 

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