Macromolecular structure phasing by neutron anomalous diffraction

Abstract

In this report we show for the first time that neutron anomalous dispersion can be used in a practical manner to determine experimental phases of a protein crystal structure, providing a new tool for structural biologists. The approach is demonstrated through the use of a state-of-the-art monochromatic neutron diffractometer at the Institut Laue-Langevin (ILL) in combination with crystals of perdeuterated protein that minimise the level of hydrogen incoherent scattering and enhance the visibility of the anomalous signal. The protein used was rubredoxin in which cadmium replaced the iron at the iron-sulphur site. While this study was carried out using a steady-state neutron beam source, the results will be of major interest for capabilities at existing and emerging spallation neutron sources where time-of-flight instruments provide inherent energy discrimination. In particular this capability may be expected to offer unique opportunities to a rapidly developing structural biology community where there is increasing interest in the identification of protonation states, protein/water interactions and protein-ligand interactions – all of which are of central importance to a wide range of fundamental and applied areas in the biosciences.

Production of perdeuterated fucose from glyco-engineered bacteria

“…2020 ), and the same is true in the case of monochromatic neutron crystallography ( Cuypers, Mason, et al. 2013a ; Cuypers et al. 2016 ).…”

“…Tailor-designed deuteration can be used very effectively in studies of multicomponent systems by small-angle neutron solution scattering (Laux et al 2008, Cuypers et al 2013b, Dunne et al 2017, Josts et al 2018, Maric et al 2019 and neutron reflection (Grage et al 2011, Hellstrand et al 2013, Waldie et al 2018. In Laue neutron crystallography, the use of perdeuterated protein imparts major benefits in terms of data quality and interpretation (Haupt et al 2014, Dajnowicz et al 2017, Yee et al 2019, Kwon et al 2020 and the same is true in the case of monochromatic neutron crystallography (Cuypers et al 2013a, Cuypers et al 2016. Deuteration of small biomolecules has wide application in analytical methods, with growing development for the study of metabolism (Shimba et al 1990) and in living cells imaging techniques such as Raman microscopy (Wei et al 2013).…”

Neutron scattering in the biological sciences: progress and prospects

“…Data scaling is then performed with routine protein crystallography software such as SCALEPACK (Otwinowski & Minor, 1997) or SCALA (Winn et al, 2011). New phasing methods are being developed, such as the use of anomalous dispersion to determine the experimental phases of protein crystal structures, providing a new tool for structural biologists (Cuypers et al, 2016).…”

Fifteen years of the Protein Crystallography Station: the coming of age of macromolecular neutron crystallography

“…Neutron diffraction data from perdeuterated and selectively deuterated protein samples have been collected at LADI-III at the ILL (Blakeley et al, 2010) and on the monochromatic instrument D19 (Cuypers et al, 2013(Cuypers et al, , 2016Haupt et al, 2014), demonstrating that deuteration can clearly shorten data-collection times, reduce the size of suitable crystals and increase the visibility of H atoms. The protocols and methods employed to deuterate proteins are well documented, have led to successful structure determinations of otherwise difficult targets and have become an essential part of neutron sources worldwide (Hazemann et al, 2005;Petit-Haertlein et al, 2009, 2010Tomanicek et al, 2011;Howard et al, 2011Howard et al, , 2016Munshi et al, 2012;Cuypers et al, 2013Cuypers et al, , 2016Meilleur et al, 2013;Weber et al, 2013;Haupt et al, 2014;Gerlits et al, 2016;Haertlein et al, 2016). In addition, computational tools were developed to simplify and integrate neutron crystallography into available programs and software suites for streamlined macromolecular crystallography (much of it developed at other DOE facilities).…”