BS3 crosslinking of GRC3 and Las1
Ribosome assembly is a complex process involving the coordination of multiple enzymes. A recently discovered endoribonuclease (RNase), Las1, and the poly-nucleotide kinase (PNK), Grc3, assemble into a complex. To attempt to understand the coordination of the two enzymes in this complex, we performed chemical crosslinking and mass spectrometry and also solved a series of cryo-electron microscopy structures of RNase PNK in multiple conformational states.
Sample Processing Protocol
CtLas1-Grc3 (10 microM) was cross-linked with 50 microM bis(sulfosuccinimidyl)suberate (BS3; Sigma) in 20 mM Hepes pH 7.7, 200 mM NaCl, 5 mM MgCl2, 5% glycerol at room temperature for 5 minutes before quenching with 30 mM Tris pH 7.5 for 15 minutes at 4C. 10 microL of the cross-linked mixture was digest by the addition of 10 microL trypsin/LysC mix (0.1 microg/microL - Promega) overnight at 40degrees C. The digests were then stored at -80degrees C for subsequent MS analysis. Protein digests were analyzed by LC/MS on a Q Exactive Plus mass spectrometer (ThermoFisher Scientific) interfaced with an M-Class nanoAcquity UPLC system (Waters Corporation) equipped with a 75 m x 150 mm BEH C18 column (1.8 m particle, Waters Corporation) and a C18 trapping column (180 m × 20 mm) with 5 m particle size at a flow rate of 400 nL/min. The trapping column was in-line with the analytical column and upstream of a micro-tee union which was used for venting, trapping, and as a liquid junction. Trapping was performed using the initial solvent composition. 5 L of digested sample was injected onto the column. Peptides were eluted by using a linear gradient from 99% solvent A (0.1% formic acid in water (v/v)) and 1% solvent B (0.1% formic acid in acetonitrile (v/v)) to 40% solvent B over 70 minutes. For the mass spectrometry, a top-ten data dependent acquisition method was employed with a dynamic exclusion time of 15 seconds and exclusion of singly charged species. The mass spectrometer was equipped with a nanoflex source with a stainless-steel needle and used in the positive ion mode. Instrument parameters were as follows: sheath gas, 0; auxiliary gas, 0; sweep gas, 0; spray voltage, 2.7 kV; capillary temperature, 275C; S-lens, 60; scan range (m/z) of 375 to 1500; 1.6 m/z isolation window; resolution: 70,000 (MS), 17,500 (MS/MS); automated gain control (AGC), 3 × 10e6 ions (MS), 5 x 10e4 (MS/MS); and a maximum IT of 100 ms (MS), 50 ms (MS/MS). Mass calibration was performed before data acquisition using the Pierce LTQ Velos Positive Ion Calibration mixture (ThermoFisher Scientific).
Data Processing Protocol
The LC/MS raw data were first converted to an MGF format using Mascot Distiller from Matrix Science and then analyzed using the Batch-Tag Web function of the ProteinProspector web-based software developed by the UCSF Mass Spectrometry Facility. The MGF file was searched against sequences for the recombinant CtGrc3 and CtLas1 by employing the User Protein Sequence field with other search parameters including: tryptic specificity and 3 missed cleavages; precursor charge range of 2, 3, 4, and 5; monoisotopic values; parent mass tolerance of 10 ppm and fragment mass tolerance of 50 ppm; oxidation of methionine as a variable modification; and in the Crosslinking field, the Link Search Type was defined as DSS. The putative cross-linked peptide output was triaged by limiting the mass error of putative cross-links to two standard deviations from the average error (about 4 ppm); requiring a Score Difference value >5 except for the cases of intermolecular cross-links of identical peptides; total expectation values below 1x10-4 with each peptide from the cross-link having expectation values below 0.1.
Jason Williams, National Institute of Environmental Health Sciences
Jason G. Williams, Mass Spectrometry Research and Support Group Epigenetics and Stem Cell Biology Laboratory National Institute of Environmental Health Sciences 111 TW Alexander Drive MD F0-04 Research Triangle Park, North Carolina 27709 ( lab head )
Pillon MC, Hsu AL, Krahn JM, Williams JG, Goslen KH, Sobhany M, Borgnia MJ, Stanley RE. Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex. Nat Struct Mol Biol. 2019 26(9):830-839 PubMed: 31488907