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. 2022 Sep 6;11:e77877. doi: 10.7554/eLife.77877

Figure 3. Crystal structure of KLP-12-tubulin complex.

(A) The steady-state ATPase activity of KLP-12(M) measured with GDP tubulin heterodimers, GTP tubulin heterodimers, and microtubules at 30 °C. Error bars represent standard deviation. Tubulin or microtubule GTPase effect was canceled by subtracting control without KLP-12(M). (B) Size exclusion chromatography (SEC) of tubulin mixed with KLP-12(M) and DARPin. (C) SDS–PAGE analysis of the SEC peaks of tubulin mixed with KLP-12(M) and DARPin. SDS-PAGE analysis of the SEC peaks of tubulin combined with KLP-12–DARPin is available in Figure 3—figure supplement 2. (D) Crystal structure of the tubulin–KLP-12–DARPin complex. Disordered linkers were drawn as a dotted line. α-tubulin is colored in light green, β-tubulin is in light blue, DARPin is in yellow, and KLP-12 is in magenta. See also Video 1 for details. (E) KLP-12 structure viewed from the interface with tubulin. The residues of the important structure are shown in color. β5a-L8-β5b is green, switch I is yellow, switch II is blue, α6 is red, and the neck linker is cyan. (F) Nucleotide binding pocket of KLP-12. The 2Fo-Fc map around AMP-PNP was calculated with coefficient 2Fo− Fc and contoured at 2.0 σ.

Figure 3—source data 1. The ATPase activity of KLP-12(M) with microtubule.
elife-77877-fig3-data1.xlsx (184.4KB, xlsx)
Figure 3—source data 2. The ATPase activity of KLP-12(M) with GTP tubulin.
elife-77877-fig3-data2.xlsx (184.2KB, xlsx)
Figure 3—source data 3. The ATPase activity of KLP-12(M) with GDP tubulin.
Figure 3—source data 4. The ATPase activity of KLP-12(M).
Figure 3—source data 5. SDS–PAGE gel of the SEC peaks of tubulin mixed with KLP-12(M) and DARPin.

Figure 3.

Figure 3—figure supplement 1. ATPase activity of KLP-12–LZ–GFP with microtubule or GTP-Tubulin.

Figure 3—figure supplement 1.

The steady-state ATPase activity of KLP-12–LZ–GFP was measured with microtubules or GTP tubulin heterodimers at 30 °C. Error bars represent standard deviation. KM,microtubules and KM,GTP-tubulin was 0.272 μM and 0.753 μM,.
Figure 3—figure supplement 1—source data 1. The ATPase activity of KLP-12–LZ–GFP with microtubule.
Figure 3—figure supplement 1—source data 2. The ATPase activity of KLP-12–LZ–GFP with GTP tubulin.
Figure 3—figure supplement 2. The complex formation of KLP-12–DARPin and tubulin dimers.

Figure 3—figure supplement 2.

(A) Size exclusion chromatography (SEC) of tubulin mixed with KLP-12–DARPin. (B) SDS-PAGE analysis of the SEC peaks.
Figure 3—figure supplement 2—source data 1. SDS-PAGE gel of the SEC peaks of tubulin mixed with KLP-12–DARPin.