Computational protocol: Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction

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Protocol publication

[…] Several crystallization conditions were tested by using the sitting-drop vapor-diffusion method with the aid of an Oryx8 robotic nanodrop dispensing system (Douglas Instruments, UK). The commercial kits JCSG+ HT96 (Molecular Dimensions, UK), Crystal Screen, PEG/Ion (Hampton Research, California, USA), and an in-house screen (80 factorial) were used for the screening. 1 µl drops of 12.5, 25 and 45 mg ml−1 RfCohScaB5-DocScaA were mixed with 1 µl reservoir solution at room temperature. The resulting plates were then stored at 292 K. Crystal formation was observed under 2 conditions (0.1 M HEPES pH 7.5, 1.2 M sodium citrate; 2.1 M DL-malic acid pH 7.0) after a period of approximately 180 days from setting up the plates (maximum dimensions ~50 × 50 × 20 μm). These crystals were cryoprotected with mother solution containing 20–30% glycerol and flash-cooled in liquid nitrogen. Preliminary X-ray diffraction experiments revealed that these crystals were of very poor quality mainly due to high mosaicity. Optimization plates based on the 2 original hits were set up. Two additive plates (one for each original condition) were also set up using the HT Additive Screen (Hampton Research, California, USA). The additive screen drops consisted of 0.8 µl protein +0.8 µl optimization condition +0.2 µl stock additive solution. This approach generated several good quality crystals. X-ray diffraction data were collected on beamline PROXIMA-1 at the Soleil Synchrotron, Saint-Aubin, France using a PILATUS 6M detector (Dectris Ltd) from crystals cooled to 100 K with a Cryostream (Oxford Cryosystems Ltd). A systematic grid search was carried out on all of these crystals to select the best diffracting part of each crystal. EDNA and iMosflm were used for strategy calculation during data collection. All data sets were processed using the Fast_dp and xia2 packages, which use the programs XDS, POINTLESS and SCALA from the CCP4 suite. Data-collection statistics are given in Table .The best diffracting crystal was formed in one of the additive screen conditions (0.1 M HEPES pH 7.5, 1.2 M Sodium Citrate, 4% v/v acetonitrile). It diffracted to a resolution of 1.4 Å and belonged to the monoclinic spacegroup P21. Phaser MR was used to carry out molecular replacement. The best solution was found using a cohesin from R. flavefaciens strain 17 ScaB (unreleased) and an ensemble of 3 R. flavefaciens FD-1 dockerins (Doc1a from 5M2O, Doc1b from 5M2S and Doc3 from 5LXV) produced with Dali. The cohesin had a sequence identity of 33.0% and the dockerins between 22% (Doc3) and 34% (Doc1b). Two copies of the heterodimer RfCohScaB5-DocScaA complex were present in the asymmetric unit. The partially obtained model was completed with Buccaneer and with manual modeling in COOT. It was then refined using REFMAC5 and PDB REDO interspersed with model adjustment in COOT. The final round of refinement was performed using the TLS/restrained refinement procedure using each module as a single group, giving the final model (Protein Data Bank code 5N5P, Table ). The root mean square deviation of bond lengths, bond angles, torsion angles and other indicators were continuously monitored using validation tools in COOT and MOLPROBITY. A summary of the refinement statistics is provided in Table . […]

Pipeline specifications

Software tools iMosflm, xia2, CCP4, PHENIX, DALI, Buccaneer, REFMAC5, MolProbity
Databases PDB_REDO
Applications Small-angle scattering, Protein structure analysis
Organisms Ruminococcus flavefaciens, Bacteria