Micromax 003

The SAXS data were collected using a pinhole camera (MolMet, Rigaku, Japan, modified by SAXSLAB/Xenocs) on a microfocused X-ray beam generator (Rigaku MicroMax 003) operating at 50 kV and 0.6 mA (30 W). Vacuum version of a Pilatus 300 K detector was used with the camera. Primary beam position and sample-to-detector distances were calibrated using an AgBehenate sample. Borosilicate capillaries were used as measurement containers for the samples. To cover the range of scattering vectors (q) from 0.005 to 1.100 Å⁻¹, two experimental settings were used.
$q=\frac{4\pi }{\lambda }\sin \theta$
where 2 $\theta$ is the scattering angle, and $\lambda$ is the incident wavelength.
We chose 22 °C and 40 °C as the measurement temperatures to obtain data throughout the thermally induced self-assembly process. Obtained scattering data were reduced using software based on the PyFAI Python library [42 (link)], and SASFit [43 (link)] and SASView [44 (link)] were used to fit the data.

TLC was performed on silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Compounds were visualized by irradiation with UV light or by treatment with 0.05 g/mL ninhydrin in ethanol or potassium iodide reagent. ¹H-NMR and ¹³C-NMR were recorded on a Bruker Avance-400 MHz instrument (Bruker BioSpin AG, Fällanden, Switzerland). HRMS were obtained with a LC-ESI-Q-TOF-MS apparatus (Waters, Milford, MA, USA). HPLC analysis of GLYX-13 was performed on an Agilent LC 1100 system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a diode array detector. Chiral analysis of intermediate were carried out using a Shimadzu LC 20AD system (SIMADZU, Kyoto, Japan) with a SPD-20A UV detector or an Agilent LC 1100 system equipped with a diode array detector. Single crystal diffraction analysis of 7 was performed on a MicroMax-003 (Rigaku Corporation, Tokyo, Japan) X-ray single crystal diffractometer and powder diffraction analysis of 7 was performed by a Bruker D8 FOCUS powder diffractometer (Bruker BioSpin AG, Fällanden, Switzerland). The L-amino acids (protected or free) were obtained from GL Biochem Ltd. (Shanghai, China). Other reagents were provided by Aladdin (Shanghai, China). The organic solvents were commercially available products (Lingfeng Chemical Reagent Co., Ltd., Shanghai, China) and were used without further purification.

Wide-angle X-ray scattering (WAXS) experiments were performed using a pinhole camera (MolMet, Rigaku, Japan, modified by SAXSLAB/Xenocs) attached to a microfocused X-ray beam generator (Rigaku MicroMax 003) operating at 50 kV and 0.6 mA (30 W). The camera was equipped with a vacuum version of Pilatus 300K 2-dimensional detector. Calibration of primary beam position and sample-to-detector distances was performed using Si powder sample. Experimental setups covered the scattering vector, q = (4π/λ)sinΘ, where λ is the wavelength and 2Θ is the scattering angle, in the range 0.22–1.42 Å⁻¹. Homemade software based on PyFAI Python library (doi:10.1088/1742-6596/425/20/202012) was used for data reduction. Samples were measured in reflection mode with incident beam angle set to 1.3 degree.

Unlabeled 14-3-3.12 was soaked into preformed crystals of 14-3-3σΔc, which grew in 25% PEG400, 5% Glycerol, 0.2 M CaCl2, 0.1 M HEPES pH 7.5 plus 2 mM BME within two weeks. The soaked crystal was fished after 15 days of incubation and flash-frozen in liquid nitrogen. Diffraction data was collected at 100K on an in-house Rigaku Micromax-003 (Rigaku Europe, Kemsing Sevenoaks, UK) sealed tube X-ray source and Dectris Pilatus 200K detector (DECTRIS Ltd Baden-Daettwil, Switzerland). Integration, scaling and merging of data was done using DIALS (CCP4i2) after which molecular replacement is done with MOLREP (CCP4i2) using PDB 4JC3 as search model. A three-dimensional structure of 14-3-3.12 was generated using eLBOW (Phenix) after which it was built within this structure based on visual inspection of Fo-Fc and 2FoFc electron density maps in Coot. Several rounds of model building and refinement (based on isotropic b-factors and standard set of stereo-chemical restraints: covalent bonds, angles, dihedrals, planarities, chiralities, non-bonded) were performed using Coot and Phenix.refine. See Supplementary Table 12 for data collection and refinement statistics. Structural coordinates have been deposited in the PDB (6TCH).