Computational protocol: Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

Similar protocols

Protocol publication

[…] Synchrotron SAXS data were collected at the Diamond Light Source on beamline B21 and the SWING beamline at SOLEIL. The Diamond data were recorded on a Pilatus 2M detector with a fixed camera length of 3.9 m and 12.4 keV energy allowing the collection of a momentum transfer range, q between 0.015 and 0.3 Å−1. All samples were extensively dialysed against the background buffer and measured for at least three construct dilutions in the concentration range that gave a monodisperse molecular weight according to SEC‐MALLS. The data at SOLEIL were recorded over a momentum transfer range of 0.01–0.43 Å−1. The two purified TRIM RBCC constructs (2.5 mg/ml TRIM25 and 6 mg/ml TRIM32) were injected onto an SEC‐3 300 Å Agilent column and eluted at a flow rate of 0.2 mg/ml at 15°C. Frames were collected continuously during the fractionation of the proteins. Frames collected before the void volume were averaged and subtracted from the signal of the elution profile to account for background scattering. Data reduction, subtraction and averaging were performed using the software FOXTROT (SOLEIL).The scattering curves were analysed using the programs Scatter and the package ATSAS to obtain the radius of gyration (R g), the maximum particle dimension (D max), the excluded particle volume (V p), the cross‐sectional radius and the pair distribution function (P(r)) (Petoukhov et al, ). The molecular mass of the scattering particles was estimated using a method described by Rambo (Rambo & Tainer, ). Low‐resolution three‐dimensional ab initio models for the different constructs of TRIM25 and 32 (with applied P2 symmetry for the dimeric and tetrameric constructs) were generated by program DAMMIF, averaging the results of 25 independent runs using the programs SUPCOMB and DAMAVER (Franke & Svergun, ). The SAXS‐derived dummy atoms models were rendered with PyMOL (Schrödinger, LLC). [...] Data for TRIM25R:UBE2D1‐Ub crystals were collected on beamline IO4 (λ = 1.2829 Å) at the Diamond Light Source (Oxford, UK) and processed using XDS. Data for TRIM32 RING domain crystals were collected on beamline ID29 at the ESRF Synchrotron Radiation Facility (Grenoble, France) at a wavelength of 1.28254 Å and processed using XDS (Kabsch, ). Both structures were solved by single‐wavelength anomalous dispersion phasing using the Zn2+ atom. Heavy‐atom search, density modification and initial model building were performed using Phenix AutoSol (Adams et al, ). Models were iteratively improved by manual building in Coot and refined using REFMAC5 and Phenix (Murshudov et al, ; Emsley & Cowtan, ). The stereochemistry of the final models was analysed with Procheck. There is no electron density for amino acids 55–58 in both TRIM32 monomers and amino acids 70–74 in one monomer. All structural figures were prepared in PyMOL.Coordinates and structure factors were deposited in the Protein Data Bank under accession codes 5FER and 5FEY. […]

Pipeline specifications