Time-resolved cryo-EM using Spotiton

Dandey, Venkata P.; Budell, William C.; Wei, Hui; Bobe, Daija; Maruthi, Kashyap; Kopylov, Mykhailo; Eng, Edward T.; Kahn, Peter; Hinshaw, Jenny E.; Kundu, Nidhi; Nimigean, Crina M.; Chen, Fan; Sukomon, Nattakan; Darst, Seth A.; Saecker, Ruth M.; Chen, James; Malone, Brandon; Potter, Clinton S.; Carragher, Bridget

doi:10.1038/s41592-020-0925-6

Cited by 121 publications

(88 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Recent evidence from biophysical studies suggests that ligands not only modulate the engagement of transducers but also the conformational ensemble of assembled receptor-transducer complexes, resulting in different signaling outcomes. To better understand the molecular basis of receptormediated effector activation and its regulation by different ligands, new technological developments are needed, such as time-resolved cryo-EM (trEM) [187][188][189][190], which will allow structural characterization of transient states that most likely play important roles for the productive engagement and subsequent dissociation of GPCR-transducer complexes. A current limitation of time-resolved cryo-EM, however, is the several millisecond-long dead time that includes the time required for sample mixing, sample delivery onto grids (e.g., by spraying) and sample vitrification.…”

Section: Concluding Remarks and Future Perspectivesmentioning

confidence: 99%

The role of structural dynamics in GPCR‐mediated signaling

Hilger

2021

The FEBS Journal

View full text Add to dashboard Cite

G protein-coupled receptors (GPCRs) play critical roles in the regulation of human physiology in response to a wide array of different extracellular stimuli and thus represent one of the largest groups of therapeutic drug targets. Recent advances in the structural characterization of GPCRs in different conformations and in complex with G proteins and arrestins have provided important insights into the mechanism and function of GPCRs. However, in order to truly understand the molecular basis of the functional versatility of GPCRs, the structural snapshots obtained by X-ray crystallography or cryo-EM need to be complimented with information about the conformational dynamics of receptors and their signaling complexes. In the last decade, a combination of biophysical approaches and computational studies has been utilized to examine the molecular motions of GPCRs and their transducer complexes and how they are regulated by ligands of different efficacy and bias. These studies revealed that GPCRs are highly dynamic allosteric proteins that can sample multiple conformational states. Ligands with distinct signaling profiles not only impact the conformational landscape of GPCRs but also of the receptor-engaged G proteins and arrestins. The conformational dynamics of GPCRs and their signaling complexes and the ligand-dependent bias sampling of distinct functional states are important underlying principles behind the complex signaling behavior of GPCRs.

show abstract

Section: Concluding Remarks and Future Perspectivesmentioning

confidence: 99%

The role of structural dynamics in GPCR‐mediated signaling

Hilger

2021

The FEBS Journal

View full text Add to dashboard Cite

show abstract

“…Carragher's team and collaborators have also imaged transient states of an ion channel, dynamin, and the ribosome (ref. 8 , this issue).…”

Section: Technology Feature Spot It Onmentioning

confidence: 97%

Molecular motion on ice

Dance¹

2020

Nat Methods

View full text Add to dashboard Cite

“…New technologies are emerging that rapidly freeze samples on timescales that prevent some of the sample from reaching and/or equilibrating at the air-water interface, in some cases improving angular distributions and reducing particle denaturation (Dandey et al, 2018(Dandey et al, , 2020Jain et al, 2012;Klebl et al, 2020;Kontziampasis et al, 2019;Noble, Wei et al, 2018;Ravelli et al, 2020). While under development to accelerate vitrification times, these technologies may prove advantageous over the use of detergents and graphene surfaces, which do not eliminate all surface interactions within the sample.…”

Section: Discussionmentioning

confidence: 99%

Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions

Atherton¹,

Moores²

2021

Acta Cryst Sect D Struct Biol

View full text Add to dashboard Cite

Kinesin-binding protein (KBP) is an important selective inhibitor of specific kinesin family members and its genetic disruption causes Goldberg–Shprintzen syndrome. Cryo-electron microscopy (cryo-EM) has recently been used to reveal the structure of KBP alone (72 kDa) and in complex with the motor domain of the mitotic kinesin-12 KIF15 (110 kDa). KBP is an α-solenoid, tetratricopeptide-repeat protein that interacts with the microtubule-binding region of the kinesin motor domain and blocks microtubule attachment. Numerous challenges arose relating to the behavior of KBP and KBP–kinesin complexes during cryo-EM sample preparation. These included the partial denaturation of KBP by air–water interfaces, protein aggregation resulting from carbon interaction and preferential orientation. Sample preparation with a graphene oxide substrate enabled the eventual structure determination. Here, experiences with preparing these samples are detailed, bringing attention to some of the challenges and opportunities that are likely to arise from protein-surface interactions.

show abstract

Time-resolved cryo-EM using Spotiton

Cited by 121 publications

References 39 publications

The role of structural dynamics in GPCR‐mediated signaling

The role of structural dynamics in GPCR‐mediated signaling

Molecular motion on ice

Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions

Contact Info

Product

Resources

About