Strategies for Automated CryoEM Data Collection Using Direct Detectors

Cheng, Anchi; Tan, Yong Zi; Dandey, Venkata P.; Potter, C. S.; Carragher, Bridget

doi:10.1016/bs.mie.2016.04.008

Cited by 21 publications

(16 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Finally, as the particle size and resolution limitations of single-particle cryo-electron microscopy (cryo-EM) continue to improve, that technology will have a major impact on drug discovery. 62 High-throughput and automation approaches, 63 combined with the size of the complexes in cryo-EM structure determination, will soon turn careful modeling of protein and ligand structural heterogeneity into a major bottleneck. qFit-ligand can provide an efficient, automated modeling approach at the amino-acid length-scale, as EM maps are immutable during modeling and refinement.…”

Section: Discussionmentioning

confidence: 99%

qFit-ligand Reveals Widespread Conformational Heterogeneity of Drug-Like Molecules in X-Ray Electron Density Maps

et al. 2018

View full text Add to dashboard Cite

Proteins and ligands sample a conformational ensemble that governs molecular recognition, activity, and dissociation. In structure-based drug design, access to this conformational ensemble is critical to understand the balance between entropy and enthalpy in lead optimization. However, ligand conformational heterogeneity is currently severely underreported in crystal structures in the Protein Data Bank, owing in part to a lack of automated and unbiased procedures to model an ensemble of protein-ligand states into X-ray data. Here, we designed a computational method, qFit-ligand, to automatically resolve conformationally averaged ligand heterogeneity in crystal structures, and applied it to a large set of protein receptor-ligand complexes. In an analysis of the cancer related BRD4 domain, we found that up to 29% of protein crystal structures bound with drug-like molecules present evidence of unmodeled, averaged, relatively isoenergetic conformations in ligand-receptor interactions. In many retrospective cases, these alternate conformations were adventitiously exploited to guide compound design, resulting in improved potency or selectivity. Combining qFit-ligand with high-throughput screening or multi-temperature crystallography could therefore augment the structure-based drug design toolbox.

show abstract

Section: Discussionmentioning

confidence: 99%

qFit-ligand Reveals Widespread Conformational Heterogeneity of Drug-Like Molecules in X-Ray Electron Density Maps

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Finally, as the particle size and resolution limitations of single-particle cryo-electron microscopy (cryo-EM) continue to improve, that technology will have a major impact on drug discovery. 59 High-throughput and automation approaches, 60 combined with the size of the complexes in cryo-EM structure determination, will soon turn careful modeling of protein and ligand structural heterogeneity into a major bottleneck. qFit-ligand can provide an efficient, automated modeling approach at the amino-acid length-scale, as EM maps are immutable during modeling and refinement.…”

Section: Discussionmentioning

confidence: 99%

qFit-ligand reveals widespread conformational heterogeneity of drug-like molecules in X-ray electron density maps

Zundert

Hudson

Keedy

et al. 2018

Preprint

View full text Add to dashboard Cite

Proteins and ligands sample a conformational ensemble that governs molecular recognition, activity, and dissociation. In structure-based drug design, access to this conformational ensemble is critical to understand the balance between entropy and enthalpy in lead optimization. However, ligand conformational heterogeneity is currently severely underreported in crystal structures in the Protein Data Bank, owing in part to a lack of automated and unbiased procedures to model an ensemble of protein-ligand states into X-ray data. Here, we designed a computational method, qFit-ligand, to automatically resolve conformationally averaged ligand heterogeneity in crystal structures, and applied it to a large set of protein receptor-ligand complexes. We found that up to 29% of a dataset of protein crystal structures bound with drug-like molecules present evidence of unmodeled, averaged, relatively isoenergetic conformations in ligand-receptor interactions. In many retrospective cases, these alternate conformations were adventitiously exploited to guide compound design, resulting in improved potency or selectivity. Combining qFit-ligand with highthroughput screening or multi-temperature crystallography could therefore augment the structurebased drug design toolbox.

show abstract

“…This is because even the best mechanical stages on the most commonly used electron microscopes can only achieve a reproducibility of +/− 1 μm when requested to move to a target some distance away (e.g. 20 μm) (Cheng et al, 2016). Alternatively, the high precision obtained by some mechanical or piezo-driven stages requires long relaxation times to reduce residual stage drift.…”

Section: Introductionmentioning

confidence: 99%

High resolution single particle cryo-electron microscopy using beam-image shift

Cheng

Alink

Rice

et al. 2018

Journal of Structural Biology

123

View full text Add to dashboard Cite

Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images. For optimal efficiency, automated data acquisition software packages typically employ some beam-image shift targeting as this method is both fast and accurate (±0.1 µm). In contrast, using only stage movement, relocation to a targeted area under low-dose conditions can only be achieved in combination with multiple iterations or long relaxation times, both reducing efficiency. Nevertheless it is well known that applying beam-image shift induces beam-tilt and with it a potential structure phase error with a phase error π/4 the highest acceptable value. This theory has been used as an argument against beam-image shift for high resolution data collection. Nevertheless, in practice many small beam-image shift datasets have resulted in 3D reconstructions beyond the π/4 phase error limit. To address this apparent contradiction, we performed cryo-EM single-particle reconstructions on a T20S proteasome sample using applied beam-image shifts corresponding to beam tilts from 0 to 10 mrad. To evaluate the results we compared the FSC values, and examined the water density peaks in the 3D map. We conclude that the phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit.

show abstract

Strategies for Automated CryoEM Data Collection Using Direct Detectors

Cited by 21 publications

References 23 publications

qFit-ligand Reveals Widespread Conformational Heterogeneity of Drug-Like Molecules in X-Ray Electron Density Maps

qFit-ligand Reveals Widespread Conformational Heterogeneity of Drug-Like Molecules in X-Ray Electron Density Maps

qFit-ligand reveals widespread conformational heterogeneity of drug-like molecules in X-ray electron density maps

High resolution single particle cryo-electron microscopy using beam-image shift

Contact Info

Product

Resources

About