Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2

Kimanius, Dari; Forsberg, Björn; Scheres, Sjors H.W.; Lindahl, Erik

doi:10.7554/elife.18722

Cited by 976 publications

(559 citation statements)

References 42 publications

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“…CTF correction, with gCTF 60 , was performed on non-dose-weighted micrographs, with all further processing on dose-weighted micrographs using RELION 2.0 61 . From 2,938 micrographs for GluA2-GSG1L bound to ZK, 541,261 particles were picked.…”

Section: Methodsmentioning

confidence: 99%

Channel opening and gating mechanism in AMPA-subtype glutamate receptors

Twomey

Yelshanskaya

Grassucci

et al. 2017

Nature

207

345

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Summary AMPA-subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission throughout the central nervous system. Gated by the neurotransmitter glutamate, AMPA receptors are critical for synaptic strength and dysregulation of AMPA receptor-mediated signaling is linked to numerous neurological diseases. Here, we use cryo-electron microscopy to solve the structures of AMPA receptor-auxiliary subunit complexes in the apo, antagonist and agonist-bound states and elucidate the iris-like mechanism of ion channel opening. The ion channel selectivity filter is formed by the extended portions of the re-entrant M2 loops, while the helical portions of M2 contribute to extensive hydrophobic interfaces between AMPA receptor subunits in the ion channel. We show how the permeation pathway changes upon channel opening and identify conformational changes throughout the entire AMPA receptor that accompany activation and desensitization. Our findings provide a framework for understanding gating across the family of ionotropic glutamate receptors and the role of AMPA receptors in excitatory neurotransmission.

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Section: Methodsmentioning

confidence: 99%

Channel opening and gating mechanism in AMPA-subtype glutamate receptors

Twomey

Yelshanskaya

Grassucci

et al. 2017

Nature

207

345

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“…CTF correction, using CTFFIND4 36 for the hTRPV6-nanodiscs data set and gCTF 37 for all other data sets, was performed on non-dose-weighted micrographs while subsequent data processing was done on dose-weighted micrographs. All other data processing, unless stated otherwise, was performed using Relion 2.0 38 . For each data set, 1000–2000 particles were manually selected to generate 2D classes for use in auto-picking.…”

Section: Methodsmentioning

confidence: 99%

Opening of the human epithelial calcium channel TRPV6

McGoldrick

Singh

Saotome

et al. 2017

Nature

194

322

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Ca2+-selective transient receptor potential vanilloid subfamily member 6 (TRPV6) channels play a critical role in calcium uptake in epithelial tissues1–4. Altered TRPV6 expression is associated with a variety of human diseases5, including cancers6. TRPV6 channels are constitutively active1,7,8 and their open probability depends on the lipidic composition of the membrane, increasing significantly in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2)7,9. We previously solved crystal structures of detergent-solubilized rat TRPV6 in the closed state10,11. Corroborating previous electrophysiological studies3, these structures demonstrated that the Ca2+ selectivity of TRPV6 arises from a ring of aspartate side chains in the selectivity filter that tightly binds Ca2+. However, it has remained unknown how TRPV6 channels open and close their pores for ion permeation. Here we present cryo-EM structures of human TRPV6 in the open and closed states. The channel selectivity filter adopts similar conformations in both states, consistent with its explicit role in ion permeation. The iris-like channel opening is accompanied by an α-to-π helical transition in the pore-lining S6 helices at an alanine hinge just below the selectivity filter. As a result of this transition, the S6 helices bend and rotate, exposing different residues to the ion channel pore in the open and closed states. This novel gating mechanism, which defines the constitutive activity of TRPV6, is unique for tetrameric ion channels and provides new structural insights for understanding their diverse roles in physiology and disease.

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“…Particles were picked from aligned dose-filtered averages using the swarm semi-automated procedure as implemented in e2boxer.py 49 , followed by extraction from unfiltered averages using Relion-2 (ref. 50 ) and local CTF parameter refinement using Gctf v1.06 (ref. 51 ).…”

Section: Methodsmentioning

confidence: 99%

The structural organization of substrate loading in iterative polyketide synthases

et al. 2018

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Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.

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Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2

Cited by 976 publications

References 42 publications

Channel opening and gating mechanism in AMPA-subtype glutamate receptors

Channel opening and gating mechanism in AMPA-subtype glutamate receptors

Opening of the human epithelial calcium channel TRPV6

The structural organization of substrate loading in iterative polyketide synthases

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