Computational protocol: A Numb–Mdm2 fuzzy complex reveals an isoform-specific involvement of Numb in breast cancer

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Protocol publication

[…] Crystallization experiments of Numb-PTBi in complex with the GPpY peptide were performed at the IEO Crystallography Unit. Diffraction-quality crystals were obtained by manual optimization of the initial conditions in hanging drops at 20°C, mixing 1 µl of protein with 0.5 µl of reservoir containing 25% PEG 3350, 0.2 M lithium sulfate, and 0.1 M Bis-Tris, pH 6. Before equilibration, drops were further supplemented with 0.5 µl of silver bullets (0.33% [wt/vol] anthrone, 0.33% [wt/vol] Congo red, 0.33% [wt/vol] N-(2-acetamido)-2-aminoethanesulfonic acid, and 20 mM Hepes, pH 6.8; n. 13) to obtain F222 crystals or with 0.5 µl of silver bullets (4 mM calcium chloride dihydrate, 4 mM magnesium chloride hexahydrate, 4 mM manganese(II) chloride tetrahydrate, 4 mM zinc chloride, and 20 mM Hepes, pH 6.8; n. 41) and 0.3 µl of additive screen (100 mM l-proline; n. 26; Hampton Research) to obtain P212121 crystals. Crystals were cryoprotected by soaking in reservoir buffer supplemented with 20% glycerol and flash-frozen in liquid nitrogen. X-ray diffraction data were collected at beamline X06DA (PXIII) of the Swiss Lightsource and beamline ID23-2 at the European Synchroton Radiation Facility. The crystals belonged to space group F222 or P212121. Initial phases were obtained by molecular replacement using the PTB domain of mouse Numb as a search model (PDB ID 3FOW). The model was then completed using iterative cycles of manual building and restrained refinement with PHENIX (). The four copies of the Numb-PTBi–GPpY complex present in the asymmetric unit (a.s.u.) of the F222 space group and the six copies of a.s.u of the P212121 space group were identical except for the insert region, which was not visible in five of the copies. Data processing and refinement statistics are shown in Table S1.For the superposition, we used the PTBi copies present in the two crystal forms in which we crystallized Numb-PTBi (P212121 and F222) and the structure of mouse PTBo (PDB ID 3F0W). The superposition was performed with the online server Rapido. [...] The images in were acquired by confocal analyses performed with a TCS SP5 acousto optical bream splitter microscope system equipped with HyD (high-quantum-efficiency hybrid detector) and photomultiplier tube detectors (Leica Microsystems). Images of the fixed samples were acquired at RT using a 40× 1.3 NA oil immersion objective under the control of LAS AF software (Leica Microsystems).The images of were acquired by widefield microscopy with a BX63 microscope equipped with an XM10 detector (Olympus). Images of the fixed samples were acquired at RT using a 20× 0.75 NA objective under the control of MetaMorph software (Molecular Devices).Images were analyzed with tools available via ImageJ/Fiji software (National Institutes of Health) and Photoshop (Adobe). […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis