Cadmium SAD phasing at CuKα wavelength

Single-wavelength anomalous diffraction (SAD) is the most common method for de novo elucidation of macromolecular structures by X-ray crystallography. It requires an anomalous scatterer in a crystal to calculate phases. A recent study by Panneerselvam et al. emphasized the utility of cadmium ions for SAD phasing at the standard synchrotron wavelength of 1 Å. Here we show that cadmium is also useful for phasing of crystals collected in-house with CuKα radiation. Using a crystal of single-domain antibody as an experimental model, we demonstrate how cadmium SAD can be conveniently employed to solve a CuKα dataset. We then discuss the factors which make this method generally applicable.


Introduction
Elucidation of atomic structures of macromolecules by X-ray crystallography requires knowledge of the phases of measured reflections. Nowadays this phase problem is most often solved by molecular replacement (MR), a computational technique which utilizes the known structure of a homologous molecule to estimate phases. However, in the case of de novo structure elucidation when an appropriate homologous structure is unavailable, phases should be determined experimentally. This is predominantly achieved by analyzing anomalous scattering produced either by atoms naturally occurring in the molecule, or intentionally introduced into crystal during growth or soaking. The two phasing methods exploiting the anomalous scattering, multiwavelength anomalous diffraction (MAD) and singlewavelength anomalous diffraction (SAD), were reviewed by Hendrickson 1 . Synchrotron radiation with tunable wavelength allows achieving the absorption edges of all elements with Z≥20 to maximize anomalous signal, thus making these methods remarkably versatile.
On the contrary, the choice of anomalous scatterer is minimal when data are to be collected in-house using a laboratory X-ray generator, most often equipped with a copper anode (λ=1.5418 Å, CuKα). Indeed, in some cases, even weak anomalous signal of sulfur (f′′=0.56eat CuKα) can be used for phasing, as demonstrated in pioneering SAD work on crambin 2 . Similarly, zinc (f′′=0.68eat CuKα) was proposed to be useful for in-house SAD experiments 3 . Perhaps the most impressive result came from the structural genomics project, where iodine ion soaks were systematically used for de novo SAD phasing of datasets collected with CuKα radiation 4 . Iodine has a strong anomalous scattering (f′′=6.9eat CuKα), high solubility, and binds multiple hydrophobic sites or positively charged residues on protein surface. Iodine SAD appeared remarkably efficient for phasing the crystals of membrane proteins which possess patches of positively charged residues at the hydrophobichydrophilic interface, providing many binding sites for anions 5 .
Another attractive opportunity is to use cadmium ions, which have a great anomalous signal (f′′=4.7eat CuKα) comparable to that of iodine, promote crystal growth 6 , and can substitute other divalent cations in metal-binding proteins. Despite all these advantages and its use in the very early SAD works 7 , Cd is rarely used in the phasing of protein crystals. Recently, a paper emphasizing the utility of cadmium ions for experimental phasing at the standard synchrotron wavelength of 1 Å was published 8 . In this short research note, we show how Cd-SAD can also be conveniently used for phasing datasets collected using CuKα radiation.

Methods
As an experimental model for in-house cadmium SAD, we used a crystal of an anti-ErbB3 single-domain antibody BCD090-M2, which we recently studied 9 . The details of protein purification, characterization, and structural analysis are given in the paper 9 . Briefly, the protein was expressed in E. coli SHuffle cells as a SUMO fusion, purified by immobilized metal affinity chromatography, cleaved by TEV protease, and then polished by an additional step of high-resolution cation-exchange chromatography. The antibody was crystallized by hanging-drop vapor diffusion in two different forms: in a space group C2 without divalent cations (PDB accession number: 6EZW) and in P1 with two cadmium ions per unit cell (PDB accession number: 6F0D) 9 . Crystals of both types diffracted below 2 Å. The data were collected on a Kappa Apex II diffractometer (Bruker AXS) using CuKα radiation generated by a IμS microfocus X-ray tube. Both structures were solved by molecular replacement in Phenix software suite v. 1.11 10 . The dataset with cadmium (6F0D) with unmerged Friedel pairs was used for SAD analysis. For experimental phasing, we used a standard protocol employing SHELXC/D/E programs 11 through HKL2MAP v. 0.4 graphical interface 12 . Data were processed with SHELXC v. 2016/1, anomalous substructure was solved by SHELXD v. 2013/2 and phasing and density modification were done by SHELXE v. 2018/2. The automatic model building and refinement were done in Phenix v. 1.14 10 , and manual refinement was done in Coot v. 0.8.9.1 13 . Figures were prepared with PyMOL.

Results and discussion
The phasing of protein crystals by SAD starts from finding the positions of an anomalous substructure, which is usually done by direct methods. First, the dataset was processed with SHELXC, and the statistical analysis of the anomalous signal is shown in Figure 1A and Table 1. The use of kappa goniometer for data collection allowed achieving high completeness (96.4%) and multiplicity (5.9) of anomalous pair measurements. The signal-to-noise ratio defined as ⟨d′′/σ(d′′)⟩ and the correlation coefficient CC 1/2 indicate that useful anomalous signal is present almost in the whole resolution range. For further substructure solution, we implied a rather conservative high-resolution cut-off of 2.4 Å corresponding to CC 1/2 (anom.) ~ 0.3.
The anomalous substructure was immediately solved by SHELXD as judged by high correlation coefficients (combined figure of merit = 55.6%), high occupancies of the two cadmium sites (1.00, 0.99), and the rapid drop in occupancy of the next site (0.17). The positions of Cd ions corresponded to the largest off-origin peak of the anomalous Patterson function at (0.58, 0.02, 0.03). The solution was used in SHELXE for phasing, electron density modification, and chain tracing. This yielded electron density maps with high contrast, and the solutions for original and inverted substructure were indistinguishable due to centrosymmetry ( Figure 1B). As discussed previously 14 , centrosymmetric anomalous sites in SAD can impede interpretation of electron density maps, because the resulting map is a superposition of the true electron density with its negative mirror-image. However, in our case the major portion of the protein chain (87%) was traced after density modification. This incomplete model was further improved in phenix.autobuild, and then refined manually in Coot and phenix.refine giving final R work /R free of 17.8/21.0%.
In this particular case, structure determination by in-house Cd-SAD was almost as straightforward as an automated molecular replacement. The causes of this simplicity were the relatively small protein size, high completeness and multiplicity of the  anomalous data, and the small number of high-occupancy cadmium sites. Furthermore, the recent theoretical study gives the following simple dependency for expected anomalous signal ⟨S ano ⟩ ~ (N refl /n sites ) 1/2 , where N refl is the number of independent reflections and n sites is the number of anomalous scatterers 15 . Our case with maximum N refl due to the lowest symmetry (P1) and only 2 anomalous sites appears virtually optimal for SAD. The high metal-binding affinity of cadmium sites was achieved through coordination with carbonyl oxygen of Glu 114 , and carboxylic groups of Asp 100 and Asp 116 ( Figure 1C). By bridging these residues to the N-terminal Gly residue of the neighboring molecule, cadmium ions effectively defined crystal contacts ( Figure 1D). Data associated with this study are available on OSF 16 .

Conclusion
In conclusion, we suggest that cadmium SAD can be generally applied for the phasing of protein crystals collected in-house using CuKα radiation. We see the following advantages of this approach: (1)  In this experiment report the authors present their work on derivatising crystals of a single-domain antibody protein with cadmium ions and subsequent solving the structure via SAD. They demonstrate that home-lab X-ray crystallographic equipment is capable of solving crystal structures via SAD at CuKalpha X-ray energy. The authors then conclude by discussing the result and suggesting the method for use in home-lab source diffraction experiments. To sum up, I find the study to be clearly presented and well prepared.
Additional notes: To my point of view, the data were collected competently to reach higher multiplicity (which is indispensable for SAD phasing) for P1 space group by exploiting kappa goniometer. The workflow for structure solution via Cd-SAD presented in this article seems to be relatively straightforward even considering the problem with centrosymmetric Cd sites (easily resolved by chain tracing). "The phasing of protein crystals by SAD starts from finding the positions of an anomalous substructure, which is usually done by direct methods." -In fact, the process involves Patterson search coupled with direct methods-based calculations namely dual-space refinement. I might suggest adding a few words on how the authors introduced cadmium ions into the crystal (I see that it was co-crystallisation as reported in their previous article).

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? 1 1.

If applicable, is the statistical analysis and its interpretation appropriate?
Not applicable

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: X-ray crystallography, small molecular and antibody therapeutic development, de novo phasing.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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