Materials and Reagents

Pipette tips (USA Scientific, TipOne, catalog number: 1111-3700)
GAC/inhibitor crystals (room temperature)

Equipment

Forceps (Fisher Scientific, catalog number: 09-753-50)
Micro tools set (Hampton Research, catalog number: HR4-811)
Pipettes (Rainin, catalog number: 17008649)
Light microscope (Olympus)
Hex key
Crystallography sample supports (MiTeGen)
1. Sample loading box: humidity controlled and includes optical microscope
2. Sample loading station: connected to a vacuum pump (controlled by a foot pedal) for sample wicking
3. Sample supports: include a frame containing the support film and a goniometer base
4. Sample Mylar seals

Software

XDS Package (https://xds.mr.mpg.de)
CCP4 Program Suite (https://www.ccp4.ac.uk)
Filtering program cut_XDS and script to run it cut_XS.csh (https://www.chess.cornell.edu/macchess/mx/mx_software)

Procedure

Handling and loading of GAC/inhibitor crystals (Figure 2)
All crystals are grown in 24-well plates at room temperature using the hanging drop vapor diffusion method. Crystals are handled in a humidity-controlled environment to help prevent crystal dehydration.
1. Assemble sample support by inserting the frame into the goniometer base and tighten with a hex key.
2. Remove a coverslip containing GAC crystals from one of the wells of the crystal tray.
3. Under the microscope and using the micro tools, pick up crystals and place them onto the film of the sample support. The landscape of the film enables the crystals to settle in random orientations, maximizing data collection. Pre-wet the film with well solution if necessary. The crystals can lie anywhere on the film.
4. After 20 or more crystals (100 × 100 × 100 μM³ in size) are on the film, transfer the sample support into the loading box and place on sample loading station.
5. Looking through the optical scope, use the foot-pedal controlled vacuum to wick away excess liquid on the sample film.
6. Remove the white covering around the sample frame (on both sides) to expose the adhesive gasket.
7. Apply the Mylar sealing film on both sides of the frame to cover and protect the crystals from drying.
8. The completed sample support containing the crystals is now ready to be mounted in the beamline hutch and exposed to X-rays.
Figure 2. Serial room temperature crystallography sample handling system. (A) Sample support containing a thin film within a rectangular frame mounted into a goniometer base. The frame is surrounded by an adhesive gasket protected by a white covering. (B) Humidity-controlled sample loading box containing an optical microscope. (C) Sample loading station that connects to a vacuum pump for liquid wicking. (D) GAC crystals on the film of the sample support.
Data collection and processing
Data were collected at the Cornell High Energy Synchrotron Source (CHESS) at Cornell University on beamline ID7B2 (FlexX).
1. Sample supports containing GAC crystals are mounted in the beamline hutch on a goniometer and raster scanned in 20 μm steps, recording 5° of oscillation at each position.
2. Each step of the raster scanning is completed in 0.75–0.5 s, with 0.25 s for data acquisition (25 frames, 0.2°, and 10 ms/frame), corresponding to a 1.3 Hz raster rate.
3. Individual oscillation frame sets are processed with XDS (processing indicated that GAC crystallized in both orthorhombic and monoclinic crystal forms. Overall, the monoclinic crystal form produced the best data quality for GAC. We selected for this polymorph by filtering the data based on unit cell dimensions and angles in XDS and reprocessed the datasets).
4. The data are then scaled using XSCALE (part of the XDS software package).
5. Datasets that poorly agree with each other are removed through filtering methods.
6. For GAC, data is filtered based on agreement of each 25-frame set with the entire set for a support, using a script (cut_XS.csh) to remove discrepant sets (with the program cut_XDS) and rescaled (with XSCALE). R-factors on intensities between an individual set and the average of all sets were used for filtering discrepant data.
7. Lists of the accepted 25-frame sets from the different sample supports are then combined using a text editor such as gedit, producing a control file for XSCALE.
8. XSCALE is then used with this control file to rescale the combined data to generate a merged dataset.
9. The merged dataset can be further filtered, as in step 6, with various values of the parameter (R-factor) defining discrepant data. The datasets are filtered in order to achieve satisfactory data completeness and statistical values.
10. Using CCP4 programs pointless, aimless, and ctruncate, the merged dataset is then converted into an mtz file for structure determination.

Acknowledgments

This work was supported by grants to R.A.C. (GM122575, CA201402), L.A.M. [University of Pittsburgh Medical Center Competitive Medical Research Fund (CMRF)], and to CHEXS (DMR-1829070), and MacCHESS (P30GM126166). We also thank MiTeGen for supplying key resources for the serial room temperature X-ray crystallography experiments. Parts of this protocol were adapted from previously described methods (Illava et al., 2021; Milano et al., 2022).

Competing interests

The authors declare that they have no conflicts of interest with the contents of this article.

References

Huang, Q., Stalnecker, C., Zhang, C., McDermott, L. A., Iyer, P., O'Neill, J., Reimer, S., Cerione, R. A. and Katt, W. P. (2018). Characterization of the interactions of potent allosteric inhibitors with glutaminase C, a key enzyme in cancer cell glutamine metabolism. J Biol Chem 293(10): 3535-3545.
Milano, S. K., Huang, Q., Nguyen, T. T., Ramachandran, S., Finke, A., Kriksunov, I., Schuller, D. J., Szebenyi, D. M., Arenholz, E., McDermott, L. A., et al. (2022). New insights into the molecular mechanisms of glutaminase C inhibitors in cancer cells using serial room temperature crystallography. J Biol Chem 298(2): 101535.
Wierman, J. L., Pare-Labrosse, O., Sarracini, A., Besaw, J. E., Cook, M. J., Oghbaey, S., Daoud, H., Mehrabi, P., Kriksunov, I., Kuo, A., et al. (2019). Fixed-target serial oscillation crystallography at room temperature. IUCrJ 6(Pt 2): 305-316.
Illava, G., Jayne, R., Finke, A. D., Closs, D., Zeng, W., Milano, S. K., Huang, Q., Kriksunov, I., Sidorenko, P., Wise, F. W., et al. (2021). Integrated sample-handling and mounting system for fixed-target serial synchrotron crystallography. Acta Crystallogr D Struct Biol 77(Pt 5): 628-644.