High resolution single particle refinement in EMAN2.1

Bell, James M.; Chen, Muyuan; Baldwin, Philip R.; Ludtke, Steven J.

doi:10.1016/j.ymeth.2016.02.018

Cited by 170 publications

(162 citation statements)

References 32 publications

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“…Per-particle alignment of the frames was performed using LM-BFGS 69 . The ~233k particle images were subjected to 2D classification in RELION to remove particles belonging to low-abundance classes and to generate projection averages for initial model generation with EMAN2 imposing C4 symmetry 70 . 181k of the particle images classified into high-abundance classes were then subjected to RELION’s 3D classification algorithm using the initial model, resulting in one major class.…”

Section: Methodsmentioning

confidence: 99%

Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel

Tao

Hite

MacKinnon

2016

Nature

210

386

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The Ca2+-activated K+ channel, Slo1, has an unusually large conductance and contains a voltage sensor and multiple chemical sensors. Dual activation by voltage and Ca2+ renders Slo1 central to numerous cellular processes that couple electrical signaling to Ca2+-mediated events such as muscle contraction and neuronal excitability. Here we present the cryo-electron microscopy structure of a full-length Slo1 channel from Aplysia californica in the presence of Ca2+ and Mg2+ at a resolution of 3.5 Å. The channel adopts an open conformation. Its voltage sensor domain adopts a non-domain-swapped attachment to the pore and contacts the cytoplasmic Ca2+-binding domain from a neighboring subunit. Unique structural features of the Slo1 voltage sensor suggest that Slo1 likely employs a voltage sensing mechanism quite distinct from known voltage-dependent ion channels. The structure reveals the molecular details of three distinct divalent cation-binding sites identified through electrophysiological studies of mutant Slo1 channels.

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Section: Methodsmentioning

confidence: 99%

Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel

Tao

Hite

MacKinnon

2016

Nature

210

386

View full text Add to dashboard Cite

show abstract

“…The initial set of 637,592 particles were subjected to 2D classification in RELION to remove particles belonging to low-abundance classes and to generate projection averages for initial model generation using EMAN2 42 . 3D classification was used to sort the remaining 453,775 particles into six classes.…”

Section: Methodsmentioning

confidence: 99%

Structural basis for gating the high-conductance Ca2+-activated K+ channel

Hite

Tao

MacKinnon

2016

Nature

172

324

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The precise control of an ion channel’s gate by environmental stimuli is crucial to the fulfillment of its biological role. The gate in Slo1 channels is regulated by two separate stimuli, intracellular Ca2+ concentration and membrane voltage. Thus, Slo1 is central to understanding the relationship between intracellular Ca2+ and membrane excitability. Here we present the Slo1 structure in the absence of Ca2+ and compare it with the Ca2+-bound channel. Ca2+ binding at two unique binding sites per subunit stabilizes an expanded conformation of the Ca2+ sensor gating ring. These conformational changes are propagated from the gating ring to the pore through covalent linkers and through protein interfaces formed between the gating ring and the voltage sensors. The gating ring and voltage sensors are directly connected through these interfaces, which allow membrane voltage to regulate gating of the pore by influencing the Ca2+ sensors.

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“…Data was binned three times. Particles were picked from the micrographs using EMAN2(Bell et al, 2016). Data was processed with RELION 1.4(Scheres, 2012).…”

Section: Star Methodsmentioning

confidence: 99%

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope

Tharakaraman

Watanabe

Chan

et al. 2018

Cell Host & Microbe

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Following the recent emergence of Zika virus (ZIKV), many murine and human neutralizing anti-ZIKV antibodies have been reported. Given the risk of virus escape mutants, engineering antibodies that target mutationally constrained epitopes with therapeutically relevant potencies can be valuable for combating future outbreaks. Here, we applied computational methods to engineer an antibody, ZAb_FLEP, that targets a highly networked and therefore mutationally constrained surface formed by the envelope protein dimer. ZAb_FLEP neutralized a breadth of ZIKV strains and protected mice in distinct in vivo models, including resolving vertical transmission and fetal mortality in infected pregnant mice. Serial passaging of ZIKV in the presence of ZAb_FLEP failed to generate viral escape mutants, suggesting that its epitope is indeed mutationally constrained. A single-particle cryo-EM reconstruction of the Fab-ZIKV complex validated the structural model and revealed insights into ZAb_FLEP’s neutralization mechanism. ZAb_FLEP has potential as a therapeutic in future outbreaks.

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High resolution single particle refinement in EMAN2.1

Cited by 170 publications

References 32 publications

Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel

Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel

Structural basis for gating the high-conductance Ca2+-activated K+ channel

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope

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