Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential

Abstract

The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e. ≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e. ≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm(3) crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H(+)) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron crystallography therefore remains the only approach where diffraction data can be collected at room temperature without radiation damage issues and the only approach to locate mobile or highly polarized H atoms and protons. Here a review of the current status of sub-atomic X-ray and neutron macromolecular crystallography is given and future prospects for combined approaches are outlined. New results from two metalloproteins, copper nitrite reductase and cytochrome c', are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 Å) X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography.

Keywords: X-ray; X-ray laser; XFEL; electron transfer; hydrogen; neutron; proton; proton coupling; protonation states; radiation damage; redox biology.

Figure 1

Neutron and joint X-ray/neutron structures of macromolecules deposited in the PDB since 2010, including data collection details and crystallographic parameters for each. The structures are ordered in terms of the ratio of the crystal volume to the asymmetric unit volume, from lowest to highest. Those with the lowest ratios can be considered the most challenging. Highlighted in red is the study of HIV-1 protease with the antiretroviral drug amprenavir bound (Weber et al., 2013 ▸) that has the lowest ratio of the crystal volume to the asymmetric unit volume. Highlighted in orange is the study of inorganic pyrophosphatase from Thermococcus thioreducens (I-PPase; Hughes et al., 2012 ▸) that currently is the largest unit cell and asymmetric unit volume to be studied. Highlighted in blue is a study of rubredoxin from Pyrococcus furiosus (RdPf; Munshi et al., 2012 ▸) that is the fastest data collection to date at 14 h. Highlighted in green is another study of RdPf (Cuypers et al., 2013 ▸) which is currently the highest resolution study at 1.05 Å. Highlighted in purple are neutron cryo-crystallography studies performed at 100 K for cytochrome c peroxidase (MW ∼34 kDa) (Casadei et al., 2014 ▸) and β-lactamase (MW ∼28 kDa) (Coates et al., 2014 ▸), and highlighted in yellow are the studies of Cu nitrite reductase (MW ∼37 kDa) from Achromobacter cycloclastes (AcNiR) and cytochrome c′ (MW ∼14 kDa) from Alcaligenes xylosoxidans (AxCytCp) presented here.

Figure 2

The PILATUS detectors have continued to develop and improve the spatial resolution, count rate, readout speed as well as sensitivity across the wavelength ranges. Most recently a unique in-vacuum X-ray detector, PILATUS 12M-DLS, has been installed on the I23 beamline at Diamond Light Source for long-wavelength X-ray crystallography. The PILATUS 12M-DLS is a semi-cylindrical detector covering a 2θ range of ±100° enabling the collection of low- and high-resolution data simultaneously. (Data were provided by Dr Clemens Schulze-Briese comparing PILATUS3 and PILATUS2.)

Figure 3

Left: the X-ray structure of AcNiR (from Achromobacter cycloclastes), showing the number of H atoms visible (1649) in the electron density maps at 0.87 Å resolution. Right: the same X-ray structure of AcNiR but showing all 2700 expected H atoms in the structure; 61% of expected H atoms are observed in this 0.87 Å resolution X-ray structure.

Figure 4

The type 2 Cu site of AcNIR with nitrite bound at 0.87 Å resolution. The catalytically important residue Asp98 is seen in multiple conformation with two distinct conformations visible. The 2F _o − F _c electron density map (in cyan) is contoured at the 1.5σ level and the F _o − F _c hydrogen omit map (in red) is contoured at the 2.0σ level.

Figure 5

Left: a neutron quasi-Laue diffraction pattern from a D-exchanged crystal of AcNiR (volume = 0.3 mm³) collected using the quasi-Laue LADI-III diffractometer. Neutron diffraction data were processed to 2.3 Å resolution. Right: zoomed-in close-up of part of the quasi-Laue diffraction pattern.

Figure 6

Large crystals of perdeuterated AcNiR have been recently grown and one of the largest (∼1 mm³) is shown here.

Figure 7

Left: the 0.84 Å resolution X-ray structure of AxCytCp, showing the number of H atoms visible (694) in the F _o − F _c hydrogen omit map (in red), contoured at the 2.0σ level. Right: the same X-ray structure of AxCytCp but showing all 989 expected H atoms in the structure.

Figure 8

The heme binding pocket of AxCytCp at 0.84 Å resolution, showing electron density for H atoms (in red) associated with key residues Leu, Phe or Met. The 2F _o − F _c electron density map (in cyan) is contoured at 1.5σ, and the F _o − F _c hydrogen omit map (in red) is contoured at 2.0σ.