We performed iterative helical real space reconstruction in RELION from BtubABC filament cryoEM images as depicted in Fig. Prosthecobacter vanneervenii PvBtubC nativeĪ: 1–473, no tag, B: 1–426 no tag, C: MGSSHHHHHH-SSGLLPRGSH-1–257ġ BtubAB heterodimer: A residues 3–435, B 2–37 46–273 281–426 Prosthecobacter vanneervenii PvBtubC EMTS derivative Encouraged by this we performed again cryoEM with subsequent 2D classification, clearly showing that BtubC binds every 8 nm, to each BtubAB heterodimer, on the outside of the filaments ( Fig. Adding more BtubC beyond the number of BtubAB heterodimers in the filaments did not significantly increase the amount of BtubC that spun down. S2) as was also reported elsewhere, recently ( 10). coli and purified with a tag, binds to BtubAB filaments in stoichiometric fashion ( Fig. Our attention turned to the third gene in the btub gene cluster, bacterial kinesin light chain, bklc ( 6), or btubC, as explained below. Because BtubA and BtubB are extremely similar in structure ( 7), we needed to add a feature to the BtubAB filaments that could mark the 8-nm heterodimer repeat along each protofilament. The alternating arrangement of BtubAB subunits means, to obtain a correct reconstruction and structure, only helical symmetry along one protofilament must be applied (twist ∼ –5.6°, rise ∼80 Å), and the reconstruction algorithm must be able to distinguish BtubA and BtubB. The approximate helical parameters as deduced from the cryoET map in C dictate that the resulting structure must have a seam where lateral interactions change from B lattice (A-A and B-B) to A lattice (A-B). ( G) Scheme summarizing the symmetry of BtubAB mini microtubules as deduced by cryoEM and cryoET: Four protofilaments with alternating filaments arrange into a hollow tube. ( F, Right) BtubC is as closely related to other TPR proteins such as MamA ( 45) as to the TPR domain of kinesin light chain ( 46). ( Inset) RELION 2D class showing the binding of BtubC every 8 nm. ( E) Transmission cryoEM image of in vitro polymerized PdBtubABC. The protein has been renamed BtubC (formerly: BKLC, bacterial kinesin light chain). dejongeii ( 6) binds stoichiometrically to BtubAB filaments. ( D) Pelleting assay showing that the third protein in the btub locus in P. ( C) Reference-free and unsymmetrised subtomogram averaged map ( 12) of BtubAB filaments showing four strands. ( B) Section through cryoET tomogram of in vitro assembled BtubAB filaments. ( Inset) RELION ( 11) 2D class compatible with four protofilaments. The filaments show helicity as indicated by crossovers (arrowheads). ( A) Transmission cryoEM image of in vitro polymerized P. BtubAB form four-stranded mini microtubules that are decorated by BtubC. Our work reveals that some bacteria contain regulated and dynamic cytomotive microtubule systems that were once thought to be only useful in much larger and sophisticated eukaryotic cells.įig. The third protein in the btub gene cluster, BtubC, previously known as “bacterial kinesin light chain,” binds along protofilaments every 8 nm, inhibits BtubAB mini microtubule catastrophe, and increases rescue. Using in vitro total internal reflection fluorescence microscopy, we show that bacterial mini microtubules treadmill and display dynamic instability, another hallmark of eukaryotic microtubules. Despite their much smaller diameter, mini microtubules share many key structural features with eukaryotic microtubules, such as an M-loop, alternating subunits, and a seam that breaks overall helical symmetry. Here, we report a 3.6-Å helical reconstruction electron cryomicroscopy structure of four-stranded mini microtubules formed by bacterial tubulin-like Prosthecobacter dejongeii BtubAB proteins. Microtubules, the dynamic, yet stiff hollow tubes built from αβ-tubulin protein heterodimers, are thought to be present only in eukaryotic cells.
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