The Neutron Macromolecular Crystallography Instruments at Oak Ridge National Laboratory: Advances, Challenges, and Opportunities

Abstract

The IMAGINE and MaNDi instruments, located at Oak Ridge National Laboratory High Flux Isotope Reactor and Spallation Neutron Source, respectively, are powerful tools for determining the positions of hydrogen atoms in biological macromolecules and their ligands, orienting water molecules, and for differentiating chemical states in macromolecular structures. The possibility to model hydrogen and deuterium atoms in neutron structures arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction studies at the 1.5–2.5 Å resolutions, which are typical for protein crystals. Neutrons have the additional benefit for structural biology of not inducing radiation damage to protein crystals, which can be critical in the study of metalloproteins. Here we review the specifications of the IMAGINE and MaNDi beamlines and illustrate their complementarity. IMAGINE is suitable for crystals with unit cell edges up to 150 Å using a quasi-Laue technique, whereas MaNDi provides neutron crystallography resources for large unit cell samples with unit cell edges up to 300 Å using the time of flight (TOF) Laue technique. The microbial culture and crystal growth facilities which support the IMAGINE and MaNDi user programs are also described.

Unusual zwitterionic catalytic site of SARS–CoV-2 main protease revealed by neutron crystallography

“…[46][47][48] Neutron diffraction data were collected using the Macromolecular Neutron Diffractometer (MaNDi) instrument at the Spallation Neutron Source (SNS). 47,[49][50][51] The crystal was held still at room temperature, and diffraction data were collected for 24 hours using all neutrons between 2-4.16 Å. Following this, the crystal was rotated by Δϕ = 10°, and a subsequent data frame was collected again for 24 hours.…”

“…The large crystal was screened for diffraction quality using a broad-bandpass Laue configuration using neutrons from 2.8 to 10 Å at the IMAGINE instrument at the high flux isotope reactor at Oak Ridge National Laboratory ( 46 , 47 , 48 ). Neutron diffraction data were collected using the macromolecular neutron diffractometer instrument MaNDi at the Spallation Neutron Source ( 47 , 49 , 50 , 51 ). The crystal was held still at room temperature, and diffraction data were collected for 24 h using all neutrons between 2 and 4.16 Å.…”

Protonation states in SARS-CoV-2 main protease mapped by neutron crystallography

“…The large crystal was screened for diffraction quality using a broad-bandpass Laue configuration using neutrons from 2.8 to 10 Å at the IMAGINE instrument at the High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory. [44][45][46] Neutron diffraction data were collected using the Macromolecular Neutron Diffractometer (MaNDi) instrument at the Spallation Neutron Source (SNS). 45,[47][48][49] The crystal was held still at room temperature, and diffraction data were collected for 24 hours using all neutrons between 2-4 Å.…”

From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography

“…This imposes limitations on the size of the crystallographic unit cell parameters, asymmetric unit volume, and it requires a very large overall crystal volume compared to what is sufficient for X-ray crystallographic studies. The general recommendation is to aim for crystals~1 mm 3 in volume with~30 out of 160 examples coming from crystals smaller than this [3,4]. A survey of deposited NPX structures in the Protein Data Bank (PDB) reveals that most large crystals were grown using vapor diffusion.…”

“…In addition to the requirement of having large, deuterated protein crystals for NPX, there are also limitations to the size of the unit cell that current neutron macromolecular beamlines can resolve. Instruments have different design features that enable some to resolve unit cell parameters of 100-150 Å on edge (IMAGINE, LADI-III, BioDIFF, iBIX), while MaNDi is designed to resolve unit cells up to 300 Å on edge [4,18]. This limitation remains a challenge, and while a large unit cell can be overcome by having a large crystal, there are very few examples of neutron structures determined from crystals with unit cells over 150 Å on any edge.…”