Journal: Current Research in Structural Biology

Article Title: Cryo-EM structure of the trehalose monomycolate transporter, MmpL3, reconstituted into peptidiscs

doi: 10.1016/j.crstbi.2023.100109

Figure Lengend Snippet: Expression, purification and reconstitution of MmpL3 Msm into peptidiscs for cryo-EM investigation A- Purification steps of MmpL3 Msm on Coomassie-stained SDS-PAGE. The shift of the protein molecular weight from ∼110 kDa to ∼85 kDa corresponds to TEV cleavage. CE, crude extract: sample after bacterial lysis obtained by sonication; SF, soluble fraction: lysed fraction after centrifugation, containing soluble proteins; CM, crude membranes: supernatant after ultracentrifugation 1 and 2; IM, insoluble membranes: resuspended pellet after ultracentrifugation 1 and 2; SM, soluble membranes: supernatant from ultracentrifugation 3, containing insoluble membranes in solution after addition of 1 % (w/v) DDM; FT, flow-through; W, wash; E, elution; NO TEV: same sample as the elution fraction (E) of IMAC 1; TEV: sample dialyzed overnight in the presence of TEV protease. B- Elution profile of MmpL3 Msm :peptidisc on size-exclusion chromatography. 1 mg of protein was incubated with the NSPr peptides at a molar ratio of 1:20. Solution was diluted to a final volume of 1 mL in buffer H and loaded on a Superdex 200 Increase 10/300 GL column and eluted in the same buffer at a flow rate of 0.35 mL min −1 on an ÄKTA pure 25 M at 4 °C. Fractions of 0.5 mL were collected. The first peak corresponds to MmpL3 Msm :peptidisc while the second corresponds to peptidiscs in excess. Fractions between the two dash lines were analyzed by Coomassie-stained SDS-PAGE. The rightmost sample corresponds to peptidisc alone, as a control. C- Representative micrograph of 3 μL of sample concentrated at 3 mg mL −1 and frozen on a holey carbon grid (Quantifoil Cu R1.2/1.3, 300 mesh).

Article Snippet: Peptidisc NSP r (amino acids sequence: N ter -FAEKFKEAVKDYFAKFWDPAAEKLKEAVKDYFAKLWD-C ter ) was obtained from Genscript with a minimum purity of 80 %.

Techniques: Expressing, Purification, Cryo-EM Sample Prep, Staining, SDS Page, Molecular Weight, Lysis, Sonication, Centrifugation, Size-exclusion Chromatography, Incubation, Control

Journal: Current Research in Structural Biology

Article Title: Cryo-EM structure of the trehalose monomycolate transporter, MmpL3, reconstituted into peptidiscs

doi: 10.1016/j.crstbi.2023.100109

Figure Lengend Snippet: Data processing, refinement and model statistics.

Article Snippet: Peptidisc NSP r (amino acids sequence: N ter -FAEKFKEAVKDYFAKFWDPAAEKLKEAVKDYFAKLWD-C ter ) was obtained from Genscript with a minimum purity of 80 %.

Techniques: Software, Biomarker Discovery

Journal: Current Research in Structural Biology

Article Title: Cryo-EM structure of the trehalose monomycolate transporter, MmpL3, reconstituted into peptidiscs

doi: 10.1016/j.crstbi.2023.100109

Figure Lengend Snippet: Cryo-EM maps quality and model building A- The local resolution map ranging from 3 to 5 Å resolution was calculated with cryoSPARC. The map attests that the highest resolution (dark blue) correlates with the transmembrane helices. The map contour (level 0.21 in ChimeraX) was adjusted so that the peptidisc peptides are not visible. B- The map at 3.2 Å (Map 2) resolution is represented as a white transparent surface. The MmpL3 Msm model fitted in the map is represented as a cartoon and colored according to the different domains. PD1 and PD2 stand for periplasmic domains 1 and 2. MMPL domain 1 encompasses the transmembrane helices 1 to 6 while MMPL domain 2 is composed of helices 7 to 12. The linker region separates the two MMPL domains. The map contour is 0.33 and displayed in ChimeraX. C- Representation of the EM map. The 3.2 Å resolution map (Map 2) is shown as a blue mesh and contoured (level 5 σ in PyMOL) around each transmembrane helix (TM) and represented as sticks. D- EM map (Map 1, level 0.12 in ChimeraX) attesting the presence of peptides from the peptidisc. The helical peptides surrounding MmpL3 Msm are shown as red cartoons while the corresponding EM map is depicted as a yellow transparent surface. Almost all the TM parts of MmpL3 Msm are embedded into peptidiscs apart from the linker region. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Peptidisc NSP r (amino acids sequence: N ter -FAEKFKEAVKDYFAKFWDPAAEKLKEAVKDYFAKLWD-C ter ) was obtained from Genscript with a minimum purity of 80 %.

Techniques: Cryo-EM Sample Prep