TakeTwo: an indexing algorithm suited to still images with known crystal parameters

Ginn, H.; Roedig, Philip; Evans, Gwyndaf; Sauter, Nicholas K.; Ernst, Oliver P.; Meents, A.; Müller‐Werkmeister, Henrike M.; Miller, Dwayne W.; Stuart, D.I.

doi:10.1107/s2053273316097084

“…For peak finding, we used peakfinder8 and min-snr = 3.5, threshold = 12. For indexing, indexers dirax 42 , xds 37 , taketwo 43 (in that order) were used, with --multi option enabled. Integration was performed using int-radius = 3,4,5.…”

Section: Methodsmentioning

confidence: 99%

Molecular mechanism of light-driven sodium pumping

Kovalev

¹

,

Astashkin

²

,

Gushchin

³

et al. 2020

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The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only nonproton cation active transporter with demonstrated potential for optogenetics. However, the existing structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools.

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“…SAD data were collected from nanocrystals soaked for 30 h with Tb-Xo4 at 10 mM in water, prior to GDVN injection 23 . Of 558747 images collected using the 5 µm beam available at LCLS-SCC, 76687, 292, 217 and 177 were indexed (i.e., a total of 77373 images) using Xgandalf 56 , Dirax 81 , taketwo 82 and Mosflm 83 , respectively, in CrystFEL v.0.8.0 57 . Post-refinement was not attempted, but images were scaled one to another using the 'unity' model in CrystFEL partialator, yielding a derivative dataset extending to 2.55 Å resolution.…”

Section: Crystal Visualization By Transmission Electron Microscopy (Tem)mentioning

confidence: 99%

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Tetreau

¹

,

Sawaya

²

,

Zitter

³

et al. 2022

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Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

show abstract

“…SAD data were collected from nanocrystals soaked for 30 hours with Tb-Xo4 at 10 mM in water, prior to GDVN injection 23 . Of 558747 images collected using the 5 μm beam available at the at LCLS-SCC, a total of 77,373 images were indexed of which 76,687, 292, 217 and 177 using Xgandalf 75 , Dirax 76 , taketwo 77 and Mosflm 78 , respectively, in CrystFEL v.0.8.0 79 . Post-refinement was not attempted, but images were scaled one to another using the ‘unity’ model in CrystFEL partialator , yielding a derivative dataset extending to 2.55 Å resolution.…”

Section: Methodsmentioning

confidence: 99%

“…Of 558747 images collected using the 5 µm beam available at the at LCLS-SCC, a total of 77,373 images were indexed of which 76,687, 292, 217 and 177 using Xgandalf 75 , Dirax 76 , taketwo 77 and Mosflm 78 , respectively, in CrystFEL v.0.8.0 79 . Post-refinement was not attempted, but images were scaled one to another using the 'unity' model in CrystFEL 1).…”

Section: Crystal Characterization By Atomic Force Microscopy (Afm) Crystals Of Cry11aa Werementioning

confidence: 99%

See 1 more Smart Citation

De novodetermination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Tetreau

¹

,

Sawaya

²

,

Zitter

³

et al. 2021

Preprint

Self Cite

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Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

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TakeTwo: an indexing algorithm suited to still images with known crystal parameters

Cited by 3 publications

References 17 publications

Molecular mechanism of light-driven sodium pumping

Molecular mechanism of light-driven sodium pumping

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

De novodetermination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

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