Cryoprobe

Optical rotations were measured using a Jasco P-2000 polarimeter. UV spectra were measured using a Beckman Coulter DU-800 spectrophotometer. ECD spectra were recorded using a Jasco J-1100 CD spectrometer, and IR spectra were recorded using a Thermo Scientific Nicolet 380 FT-IR spectrometer. NMR spectra were collected using both a Bruker 800 MHz NMR instrument equipped with a cryoprobe and a Varian 500 MHz instrument. The chemical shifts reported were referenced to the residual solvent peaks of CDCl₃ (δ_H 7.26 and δ_C 77.2). HRESIMS analysis was performed using an AB SCIEX TripleTOF 4600 mass spectrometer with Analyst TF software. LC-HRESIMS experiments for molecular networking were performed using a Thermo LTQ Orbitrap XL high-resolution ESI mass spectrometer coupled to Thermo U3000 HPLC system, equipped with a solvent reservoir, in-line degasser, binary pump and refrigerated autosampler. Low resolution LC-MS was performed using a Thermo Fisher Scientific ISQ mass spectrometer with an electrospray ionization (ESI) source. Semi-preparative HPLC was carried out using a Dionex UltiMate 3000 HPLC system equipped with a micro vacuum degasser, an autosampler, and a diode-array detector.

NMR experiments were performed on Varian direct drive 600 and 800 MHz
spectrometers equipped with a cryoprobe. Assignment of backbone atoms for
N-Cdc37 was obtained by standard triple-resonance pulse sequences, aided by
selective amino acid labeling/unlabeling and a 3D HNN experiment (Panchal et al., 2001 (link)). Sidechain and methyl
group assignment was accomplished with TOCSY-based experiments, in combination
with side-directed mutagenesis. Methyl group assignment transfer from isolated
domains to full-length Cdc37 was assisted by a 3D HMQC-NOESY-HMQC spectrum
acquired in D₂O and further aided by a series of mutants at linker
regions. All spectra were processed with NMRpipe (Delaglio et al., 1995 (link)) and analyzed using sparky (T.D. Goddard and
D.G. Kneller, SPARKY 3, University of California, San Francisco). NOE restraints
were derived from a 3D ¹⁵N-edited NOESY-HSQC and a 3D
HMQC-NOESY-HMQC. Pairs of unambiguously assigned NOEs, TALOS-derived dihedral
angles (Shen and Bax, 2013 (link)), and a set of
hydrogen bonds were incorporated as restraints in CYANA 2.1 (Güntert et al., 1997 (link)).

For the (5’-GGUGGCUGUU-3’)₂ NMR sample, the H₂O 2D NOESY and ¹⁵N HMQC spectra were collected on a Varian 800 MHz spectrometer with a cryoprobe and a 600 MHz spectrometer, respectively, at the National Magnetic Resonance Facility in Madison, Wisconsin (NMRFAM). Data processing was performed with NMRPipe³⁷ and SPARKY³⁸ . For 5’-GUGUCGGUGU-3', SNOESY, COSY, ³¹P HETCOR, ¹³C and ¹⁵N HSQC spectra were collected on a Varian 500 MHz spectrometer using Biopack software^{39 (link)}. Data processing and analysis was performed with Varian software and SPARKY³⁸ . Detailed experimental parameters are shown in Supporting Information Table 2. No imino proton resonances in the ultimate and penultimate G·U pairs for all three sequences were observed at any temperature. The lack of cross-strand NOEs for the ultimate and penultimate G·U pairs precludes rigorous structure determination by NMR constraints. Thus modelling with ROSIE, which has the ability to incorporate constraints from NMR data, was pursued rather than simulated annealing methods for NMR structure determination.