A microtubule RELION-based pipeline for cryo-EM image processing

Cook, Alexander D.; Manka, Szymon W.; Wang, Su; Moores, Carolyn A.; Atherton, Joseph

doi:10.1016/j.jsb.2019.10.004

Cited by 47 publications

(28 citation statements)

References 53 publications

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“…For the catalytically inactive mutant E254A (in α-tubulin), cryo-EM processing protocols developed for and applied to MTs ( 28 , 29 ) failed to yield a high-resolution structure. Some of these E254A MTs had an abnormally large twist parameter, a feature that was also visible in raw images and 2D (two-dimensional) class averages ( SI Appendix , Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

LaFrance

Roostalu

Henkin

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis–deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.

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Section: Resultsmentioning

confidence: 99%

Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

LaFrance

Roostalu

Henkin

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…The KIF15_MD6S-MT dataset was processed using our MT RELION-based pipeline (MiRP) as described previously, using low-pass filtered KIF5B_MD-decorated MTs as references ( Atherton et al, 2019 ; Cook et al, 2020 ). KIF15_MD6S 13-protofilament-MTs were the most common MT architecture and were selected after supervised 3D classification in MiRP for analysis.…”

Section: Methodsmentioning

confidence: 99%

The mechanism of kinesin inhibition by kinesin-binding protein

Atherton

Hummel

Olieric

et al. 2020

eLife

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Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a TPR-containing, right-handed α-solenoid that sequesters the kinesin motor domain’s tubulin-binding surface, structurally distorting the motor domain and sterically blocking its MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain, and selected structure-guided mutations disrupt KBP inhibition of kinesin transport in cells. The KBP-interacting motor domain surface contains motifs exclusively conserved in KBP-interacting kinesins, suggesting a basis for kinesin selectivity.

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“…Particles with box size of 432 pixels ( Pb kinesin-8B-MD NN), 576 pixels ( Pb kinesin-8B-MD AMPPNP, Pf kinesin-8B-MD NN) and non-overlapping region of one tubulin dimer size were picked using EMAN2 e2helixboxer.py 60 . All picked particles were imported into RELION v3.0 and performed using the MiRP pipeline 61-63 . Briefly, 4x binned particles were subjected to supervised 3D classification with 15 Å low-pass filtered references of MTs with different protofilament numbers.…”

Section: Methodsmentioning

confidence: 99%

Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission

Liu

Shilliday

Cook

et al. 2022

Preprint

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Kinesins are a superfamily of molecular motors that undertake ATP-dependent microtubule-based movement or regulate microtubule dynamics. Plasmodium species, which cause malaria and kill hundreds of thousands annually, encode 8-9 kinesins in their genomes. Of these, two - kinesin-8B and kinesin-8X - are canonically classified as kinesin-8s, which in other eukaryotes typically modulate microtubule dynamics. Unexpectedly, Plasmodium kinesin-8B is required for development of the flagellated male gamete in the mosquito host, and its absence completely blocks parasite transmission. To understand the molecular basis of kinesin-8B's essential role, we characterised the in vitro properties of the kinesin-8B motor domains from P. berghei and P. falciparum. Both motors drive plus-end directed ATP-dependent microtubule gliding, but also catalyse ATP-dependent microtubule depolymerisation. We determined the microtubule-bound structures of these motors using cryo-electron microscopy. P. berghei and P. falciparum kinesin-8B exhibit a very similar mode of microtubule interaction, in which Plasmodium-distinct sequences at the microtubule-kinesin interface influence motor function. Intriguingly, however, P. berghei kinesin-8B exhibits a non-canonical structural response to ATP analogue binding such that neck linker docking is not induced. Nevertheless, the neck linker region is absolutely required for motility and depolymerisation activities of these motors. Taken together, these data suggest that the mechanochemistry of Plasmodium kinesin-8Bs has been functionally tuned to efficiently contribute to flagella formation.

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A microtubule RELION-based pipeline for cryo-EM image processing

Cited by 47 publications

References 53 publications

Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

The mechanism of kinesin inhibition by kinesin-binding protein

Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission

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