Joint X-ray/neutron structure of <i>Lentinus similis</i> AA9_A at room temperature

Tandrup, Tobias; Leggio, Leila Lo; Meilleur, Flora

doi:10.1107/s2053230x22011335

Cited by 4 publications

(2 citation statements)

References 40 publications

(45 reference statements)

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“…2(a) shows a histogram of the author-supplied highresolution limit (d min ) of the neutron data. The best resolution is 1.05 A ˚for PDB entry 4ar3 (Cuypers et al, 2013) and the lowest resolution is 2.8 A ˚for PDB entry 8e1w (Tandrup et al, 2023). For all neutron entries the average high-resolution limit is 1.99 A ˚, while for joint XN and only neutron entries the average d min amounts to 2.04 and 1.91 A ˚, respectively.…”

Section: Overview Of Some Neutron Model and Data Propertiesmentioning

confidence: 95%

Improved joint X-ray and neutron refinement procedure in Phenix

Liebschner,

Afonine,

Poon

et al. 2023

Acta Cryst Sect D Struct Biol

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Neutron diffraction is one of the three crystallographic techniques (X-ray, neutron and electron diffraction) used to determine the atomic structures of molecules. Its particular strengths derive from the fact that H (and D) atoms are strong neutron scatterers, meaning that their positions, and thus protonation states, can be derived from crystallographic maps. However, because of technical limitations and experimental obstacles, the quality of neutron diffraction data is typically much poorer (completeness, resolution and signal to noise) than that of X-ray diffraction data for the same sample. Further, refinement is more complex as it usually requires additional parameters to describe the H (and D) atoms. The increase in the number of parameters may be mitigated by using the `riding hydrogen' refinement strategy, in which the positions of H atoms without a rotational degree of freedom are inferred from their neighboring heavy atoms. However, this does not address the issues related to poor data quality. Therefore, neutron structure determination often relies on the presence of an X-ray data set for joint X-ray and neutron (XN) refinement. In this approach, the X-ray data serve to compensate for the deficiencies of the neutron diffraction data by refining one model simultaneously against the X-ray and neutron data sets. To be applicable, it is assumed that both data sets are highly isomorphous, and preferably collected from the same crystals and at the same temperature. However, the approach has a number of limitations that are discussed in this work by comparing four separately re-refined neutron models. To address the limitations, a new method for joint XN refinement is introduced that optimizes two different models against the different data sets. This approach is tested using neutron models and data deposited in the Protein Data Bank. The efficacy of refining models with H atoms as riding or as individual atoms is also investigated.

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Section: Overview Of Some Neutron Model and Data Propertiesmentioning

confidence: 95%

Improved joint X-ray and neutron refinement procedure in Phenix

Liebschner,

Afonine,

Poon

et al. 2023

Acta Cryst Sect D Struct Biol

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“…This alleviates the sample volume requirement by as much as a factor of ten. A few neutron crystal structures of LPMOs have already been determined; − however, none of them is perdeuterated, enabling mapping of only select hydrogen atoms.…”

Section: Introductionmentioning

confidence: 99%

Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase

Sørensen,

Montserrat-Canals,

Loose

et al. 2023

ACS Omega

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Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen’s large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields >10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.

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