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.
Do you know what TANTALUM CLUSTER, TUNGSTEN CLUSTER, LANTHANIDE CARRIER, or MAGIC TRIANGLE is?
Min-Kun Kim (KIOST): Let
me introduce our recently published result in Acta D (2012)
1253~1258 entitled "Experimental phasing with zinc anomalous
scattering". (Edited by Dr. Manfred S. Weiss and this result is
presented in ECM27, Bergen, Norway in 7th Aug. 2012 by Dr.
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 (firstname.lastname@example.org).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.
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