Electron diffraction of SmNuc1 nuclease

T. Skálová1, P. Brázda2, D. G. Waterman3,4, K. Adámková1, J. Dohnálek1

1 Institute of Biotechnology of the Czech Academy of Sciences, v.v.i., Průmyslová 595, 252 50, Vestec, Czech Republic

 2 Institute of Physics, Czech Academy of Sciences, Prague, Czech Republic

3 STFC Rutherford Appleton Laboratory, Didcot, UK

4 CCP4, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK

t.skalova@gmail.com

Stenotrophomonas maltophilia is a gram-negative bacterium from the class Gammaproteobacteria, recognized as a cause of hospital-acquired infections and notable for its multidrug resistance. One of its nucleases, SmNuc1, belongs to the S1-P1 family of enzymes that cleave nucleic acids. While S1-P1 nucleases are found in several multidrug-resistant bacteria, their biological roles remain poorly understood.

SmNuc1 is a zinc-dependent enzyme with a predominantly α-helical fold. Its active site is a surface cleft containing a trinuclear zinc cluster coordinated by nine residues. Previous studies have described the expression, purification, and characterization of SmNuc1 [1], and recently, seven X-ray crystal structures — the free enzyme and complexes with RNA, cleavage products — have been reported [2].

Small needles of SmNuc1 were crystallized, pipetted to golden grids and vitrified using a Vitrobot. Electron diffraction data were collected in the Institute of Physics AS CR (a 200 kV transmission electron microscope FEI Tecnai G2 20 equipped with a CheeTah M3 hybrid pixel detector). The collected diffraction data were visually checked using PETS2 [3] and finally processed in DIALS [4]. The data merged from five crystals were processed to 2.86 Å resolution with overall completeness 83.4 %. The phase problem was solved by molecular replacement in Phaser [5] using one chain of a SmNuc1 X-ray crystal structure with PDB code 8QJO. The structure is under refinement in Phenix [6] with current R-factor 30.9 % and Rfree 35.6 %.

The presentation will describe our first protein structure solved using electron diffraction and overcoming difficulties in individual steps of solving the structure.

 

1. Husťáková, B., Trundová, M., Adámková, K., Kovaľ, T. Dušková, J., Dohnálek, J. (2023).  FEBS Lett. 597, 2103-2118.

2. Adámková, K., Trundová, M., Koval, T., Husťáková, B., Kolenko, P., Dušková, J., Skálová, T., Dohnálek, J. (2025). FEBS J. 292, 129-152.

3. Palatinus, L., Brázda, P., Jelínek, M., Hrdá, J., Steciuk, G., Klementová, M. (2019). Acta Crystallogr. B 75, 512-522.

4. Winter, G., Beilsten‐Edmands, J., Devenish, N., Gerstel, M., Gildea, R. J., McDonagh, D., Pascal, E., Waterman, D. G., Williams, B. H., Evans, G. (2022). Protein Science, 31(1), 232–250.

5. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., Read, R. J. (2007). Journal of Applied Crystallography, 40(4), 658–674.

6. Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L.-W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., Zwart, P. H. (2010). Acta Crystallographica Section D Biological Crystallography, 66(2), 213–221.

We acknowledge the core facilities of CMS-Biocev (CF Crystallisation of proteins and nucleic acids) of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2023042) and CZ.02.1.01/0.0/0.0/18_046/0015974 and IMCF-Biocev supported by MEYS CR (LM2023050 Czech-BioImaging). This work was supported by the institutional support of IBT CAS, v.v.i. (RVO: 86652036) and the Czech Science Foundation (25-17546S). Electron diffraction studies were supported by the CzechNanoLab Research Infrastructure supported by MEYS CR (LM2023051) and project Terafit supported by the MEYS CR (CZ.02.01.01/00/22_008/0004594).