Automated data collection in single particle electron microscopy

Tan, Yong Zi; Cheng, Anchi; Potter, Clinton S.; Carragher, Bridget

doi:10.1093/jmicro/dfv369

Cited by 62 publications

(47 citation statements)

References 71 publications

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“…With dose fractionation set at 1.5 s per frame, each movie series contained 38 frames and each frame received a dose of 1.05 electrons per Å 2 . Fully automated data collection was carried out using EPU software with a nominal defocus range set from −1.25 to −4 μm 45 .…”

Section: Methodsmentioning

confidence: 99%

Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin

et al. 2018

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The bacterial type III secretion system, or injectisome, is a syringe shaped nanomachine essential for the virulence of many disease causing Gram-negative bacteria. At the core of the injectisome structure is the needle complex, a continuous channel formed by the highly oligomerized inner and outer membrane hollow rings and a polymerized helical needle filament which spans through and projects into the infected host cell. Here we present the near-atomic resolution structure of a needle complex from the prototypical Salmonella Typhimurium SPI-1 type III secretion system, with local masking protocols allowing for model building and refinement of the major membrane spanning components of the needle complex base in addition to an isolated needle filament. This work provides significant insight into injectisome structure and assembly and importantly captures the molecular basis for substrate induced gating in the giant outer membrane secretin portal family.

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Section: Methodsmentioning

confidence: 99%

Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin

et al. 2018

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“…Movies (60 frames) were acquired in counting mode at a magnification of 59,000x resulting in a pixel size of 1.14Å/pix. Images were collected automatically using EPU software (Thermo Fisher Co.) within a defocus range of 1.0-2.5µm under focus 23 .…”

Section: Expression and Purification Of Trpv5mentioning

confidence: 99%

Structural insights on TRPV5 gating by endogenous modulators

Hughes

Pumroy

Yazici

et al. 2018

Preprint

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TRPV5 is a transient receptor potential channel involved in the vital process of calcium homeostasis, specifically at the level of calcium reabsorption in the kidney. TRPV5 is a constitutively open channel and numerous endogenous modulators tightly regulate calcium entry through the TRPV5 channel into the cell. Here we used cryo-electron microscopy (cryo-EM) to investigate the interaction of two such modulators with fulllength TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) and calmodulin (CaM) have been reported to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-EM, we have determined the TRPV5 structure in the presence of dioctanoyl PI(4,5)P 2 , which revealed the key role annular lipids play in maintaining the channel in a conducting state and highlighted the possibility of a transient interaction between dioctanoyl PI(4,5)P 2 and the channel. Additionally, we have uncovered the mechanism of TRPV5 calcium-dependent inactivation, which is mediated by the binding of one CaM molecule per TRPV5tetramer. This novel mechanism involves the C-lobe of calcium activated CaM physically obscuring the ion conducting pore of TRPV5 by binding to critical tryptophan residues (Trp583) at the intracellular gate of TRPV5. This interaction is initiated by the binding of the CaM C-lobe to the distal C-terminus of TRPV5 which brings CaM in close proximity to the TRPV5 pore. Overall, this investigation has provided insight into the endogenous modulation of TRPV5 which has the potential to guide in silico drug discovery and rational drug design for targeted therapeutics for a variety of calcium dependent kidney diseases.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx

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“…Several methodological challenges still prevail in all steps of atypical project workflow.Although this is atopic of intense research, the lack of automation seems to be one of its biggest issues.Currently,ahigh-resolution cryo-EM structure requires the collection of thousands of images over several days at the electron microscope.G iven that each DED "movie" is composed of several frames,complete data sets are typically in the range of several Tbytes.However,with storage space becoming very cheap,d ata collection has become one of the best automated parts of the cryo-EM process,w ith several packages able to schedule the imaging,h andle imaging conditions and acceptable image drift, and even connect to databases (for ar eview,s ee Ref. [96]). Moreover, since,h igh-end microscopes can preserve their alignment during long collection sessions,a nd their vacuums are good enough so that little ice contamination builds up,a utomated data collection has become the norm.…”

Section: Challenges In Automationmentioning

confidence: 99%

Electron Cryo‐microscopy as a Tool for Structure‐Based Drug Development

Merino

Raunser

2017

Angew Chem Int Ed

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For decades, X-ray crystallography and NMR have been the most important techniques for studying the atomic structure of macromolecules. However, as a result of size, instability, low yield, and other factors, many macromolecules are difficult to crystallize or unsuitable for NMR studies. Electron cryo-microscopy (cryo-EM) does not depend on crystals and has therefore been the method of choice for many macromolecular complexes that cannot be crystallized, but atomic resolution has mostly been beyond its reach. A new generation of detectors that are capable of sensing directly the incident electrons has recently revolutionized the field, with structures of macromolecules now routinely being solved to near-atomic resolution. In this review, we summarize some of the most recent examples of high-resolution cryo-EM structures. We put particular emphasis on proteins with pharmacological relevance that have traditionally been inaccessible to crystallography. Furthermore, we discuss examples where interactions with small molecules have been fully characterized at atomic resolution. Finally, we stress the current limits of cryo-EM, and methodological issues related to its usage as a tool for drug development.

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Automated data collection in single particle electron microscopy

Cited by 62 publications

References 71 publications

Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin

Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin

Structural insights on TRPV5 gating by endogenous modulators

Electron Cryo‐microscopy as a Tool for Structure‐Based Drug Development

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