Tips and Tricks in Crystallography - Structure Solution

FSEARCH: cryo-EM Map Replacement |  Recently, a  hybrid method that integrates X-ray crystallography with cryo-EM for structure determination is presented by Lingxiao Zeng, Wei Ding, and Quan Hao in the IUCrJ (5:382-389, 2018). The starting point is a cryo-EM map  and the end point is a high-resolution crystal structure. The workflow of the method and four case study examples can be seen here.

A new MR-SAD algorithm
Recently, a  new MR-SAD algorithm was  reported  by Pavol Skubak, Navray Pannu, and co-workers in the IUCrJ (5:166-171, 2018), for automatic building of protein models from low-resolution X-ray diffraction data and a poor starting model. According to the authors, their algorithm uses a multivariate function to simultatnerously exploit informatino from both the initial partial model and low-resolution SAD data. Details of the algorithm and its application to six challenging structure determinations can be seen here.

Zbigniew Dauter (NCI): Selenourea: A Convenient Phasing Vehicle

Majority of novel X-ray crystal structures of proteins are currently solved using the anomalous diffraction signal provided by selenium after incorporation of selenomethionine instead of natural methionine by genetic engineering methods. However, selenium can be also inserted into protein crystals in the form of selenourea (SeC(NH2)2), by adding selenourea into mother liquor or cryo-solution or in the form of powder into a drop with native crystals, in analogy to the classic procedure of heavy-atom derivatization. Selenourea is able to bind to reactive groups at the surface of macromolecules primarily through hydrogen bonds, where the selenium atom may serve as acceptor and amide groups as H-bond donors. Selenourea has different chemical properties than other heavy-atom reagents and halide ions and provides a convenient way of phasing crystal structures of macromolecules. A de novo protein crystal structure at low resolution of 2.9 Å with total 1125 residues distributed in seven chains in asymmetric unit at was recently successfully solved with this method. For details, see Selenourea: a convenient phasing vehicle for macromolecular X-ray crystal structures, Sci. Rep. 6, 37123 (2016) by Zhipu Luo.


Min-Kun Kim (KIOST): Let me introduce our recently published result in Acta D (2012) 68(Pt 9): 1253~1258 entitled "Experimental phasing with zinc anomalous scattering". (Edited by Dr. Manfred S. Weiss and this result is also presented in ECM27, Bergen, Norway in 7th Aug. 2012 by Dr. Sun-Shin Cha) 
MAD/SAD are the prevailing methods to determine novel structures. Anomalous scattering of transition metals including selenium, mercury, lead, platinum, and gold is critical for MAD and SAD. Zinc is also a suitable metal for MAD/SAD, considering the fact that structures of proteins with intrinsically-bound zinc are successfully determined by zinc anomalous scattering. However, zinc is not used for structure determination of proteins without zinc-binding sites. We noticed that proteins whose crystals were grown in crystallization reagents containing zinc acetate have zinc ions on their surface. Zinc ions are coordinated by a single amino acid (histidine, aspartate, glutamate, asparagine, or glutamine) and water molecules (or acetate molecules) with a tetrahedral or octahedral geometry, indicating that the surface of any protein can be coated with zinc ions. Based on this observation, we soaked three protein crystals in a zinc-containing solution and solved the structures by SAD, demonstrating the effectiveness of the zinc-coating method for structure determination.

We hope that you would share these ideas and get successful results in correspond cases. Please share your comments and advices anytime to Dr. Cha (

Lothar Esser (NCI): The Successor of BEAST - Molecular replacement is always a bit tricky especially with poor data. The membrane protein that I work on provides only very anisotropic data and the only program that could handle it was the successor of BEAST: Phaser. So many people have said so many good things about phaser ( see G. Sheldrick's comments on the CCP4BB ) that I do not need to add anything only so much that it solved my difficult problem too. I have a solution despite less than ideal data. What usually convinces me of the correctness of a solution is when I see peaks in the anomalous difference map for sulfur atoms or atoms of which Z>=16. Just to be on the safe side, my MR models are usually free of S or any heavy atoms so that when I get peaks in the right places, I am convinced. Coming back to data, Phaser rejects outliers also but its great strength (at least in my case) lies in the correction of anisotropic data. I'd highly recommend it.

Xinhua Ji (NCI): The Beauty of BEAST - Outliers among diffraction data are usually of lower resolution and relatively stronger and thereby have greater impact on the functions derived from intensities. One consequence of this impact is the lack of phasing power of molecular replacement (MR) solution. We recently encountered this problem with an MR solution obtained using a search model equivalent to ~50% the structure. With AMoRe, we found an solution; but we were not able to complete the structure using either the difference Fourier synthesis or other programs. Assuming that the MR solution was not accurate, we tried MR again excluding potential outliers. BEAST identified 22 potential outliers and rejected them from likelihood calculations. The MR solution was outstanding and the difference map derived from the partial structure reveals the missing portion of the structure.

Biomolecular Structure Section (NCI): SOLVE is great. It can carry out a complete and automatic structure determination for either MIR or MAD data. What if it does not work automatically or cannot find all heavy atom sites? We have experienced the following.

Case I: HgMAD data were collected at four wavelengths. Difference Patterson indicated five Hg sites. Using all wavelengths, however, SOLVE was not able to find any consistent set of Hg sites. Slight adjustment of any parameter (resolution, nres, nanomalous, etc.) resulted in a complete different set of sites. None of the sites was verifiable with difference Patterson. The last thing we tried was to feed SOLVE with different combination of wavelengths. When wavelength 1 was excluded, SOLVE found a consistent set of four correct Hg sites. Yet, one site was still missing. Then, we fed SOLVE with unmerged data, which resulted in five correct sites immediately. Guess what? When the excluded wavelength 1 for SOLVE was included in SHARP refinement, it did help improve the phases! To the best of our knowledge, nothing is significantly wrong with wavelength 1, which was collected from the same crystal and processed by the same crystallographer.

Case II: A total of 24 Se sites were expected. SOLVE found 21 using premerged data. With unmerged data, it found 25 (24 + 1 alternate position for one Se atom)!

To generate unmerged data, one needs to tell SCALEPACK “no merge original index” and then to tell SOLVE “unmerged" before running the program.

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