Xtrapol8 enables automatic elucidation of low-occupancy intermediate-states in crystallographic studies

Zitter, E. De; Coquelle, Nicolas; Oeser, Paula; Barends, Thomas R. M.; Colletier, J.P.

doi:10.1038/s42003-022-03575-7

Cited by 27 publications

(39 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Indeed, extrapolation may even introduce negative structure factor amplitudes into the data set. However, at 1.6 Å resolution less than ten percent of our extrapolated structure factors are affected by this problem and “rescuing” them using the methods recently evaluated by de Zitter and coworkers 53 did not result in appreciable improvements. We therefore did not apply these methods.…”

Section: Methodsmentioning

confidence: 76%

Influence of pump laser fluence on ultrafast structural changes in myoglobin

Barends

Bhattacharyya

Gorel

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer. However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore. As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern whether this experimental approach allows valid inferences to be drawn vis-a-vis biologically relevant single-photon-induced reactions. Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments such that biologically relevant insight emerges.

show abstract

Section: Methodsmentioning

confidence: 76%

Influence of pump laser fluence on ultrafast structural changes in myoglobin

Barends

Bhattacharyya

Gorel

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…With current advancements in data analysis ( e.g. Xtrapol8, (De Zitter et al , 2022)), it is projected that mixed diffraction data of multiple states can be separated.…”

Section: Resultsmentioning

confidence: 99%

Mix-and-extrude: high-viscosity sample injection towards time-resolved protein crystallography

Vakili

Han

Schmidt

et al. 2022

Preprint

View full text Add to dashboard Cite

Time-resolved crystallography enabled the visualization of protein molecular motion during reaction. Currently, light is the most prevalent reaction initiator, but the number of light-activated proteins is extremely limited. Instead, more biological reactions are triggered by interaction with ligands. To examine the molecular action of these mixing-initiated reactions by X-ray diffraction, a sample delivery method that allows for diffusive mixing of crystals with ligands in a short amount of time corresponding to the reaction time to be studied is needed. This paper describes the mix-and-extrude nozzle for delivering membrane proteins crystallized in lipid cubic phase and ligands, along with its preliminary characterization and outlook for future developments.Synopsis3D printed mixing-HVE devices address time-resolved membrane protein crystallography challenges via compact dual-flow LCP injection.

show abstract

“…2). Occupancy refinement is an ongoing area of research and several more rigorous methods are available to potentially tease out these subtleties (Pearce, Krojer, Bradley et al, 2017;De Zitter et al, 2022), which might be key components in biological processes such as ligand binding. Until occupancy refinement improves, the user has the final word in inter-preting the conformations in the context of the rest of the structure.…”

Section: Discussionmentioning

confidence: 99%

FLEXR: automated multi-conformer model building using electron-density map sampling

Stachowski¹,

Fischer²

2023

Acta Crystallogr D Struct Biol

View full text Add to dashboard Cite

Protein conformational dynamics that may inform biology often lie dormant in high-resolution electron-density maps. While an estimated ∼18% of side chains in high-resolution models contain alternative conformations, these are underrepresented in current PDB models due to difficulties in manually detecting, building and inspecting alternative conformers. To overcome this challenge, we developed an automated multi-conformer modeling program, FLEXR. Using Ringer-based electron-density sampling, FLEXR builds explicit multi-conformer models for refinement. Thereby, it bridges the gap of detecting hidden alternate states in electron-density maps and including them in structural models for refinement, inspection and deposition. Using a series of high-quality crystal structures (0.8–1.85 Å resolution), we show that the multi-conformer models produced by FLEXR uncover new insights that are missing in models built either manually or using current tools. Specifically, FLEXR models revealed hidden side chains and backbone conformations in ligand-binding sites that may redefine protein–ligand binding mechanisms. Ultimately, the tool facilitates crystallographers with opportunities to include explicit multi-conformer states in their high-resolution crystallographic models. One key advantage is that such models may better reflect interesting higher energy features in electron-density maps that are rarely consulted by the community at large, which can then be productively used for ligand discovery downstream. FLEXR is open source and publicly available on GitHub at https://github.com/TheFischerLab/FLEXR.

show abstract

Xtrapol8 enables automatic elucidation of low-occupancy intermediate-states in crystallographic studies

Cited by 27 publications

References 36 publications

Influence of pump laser fluence on ultrafast structural changes in myoglobin

Influence of pump laser fluence on ultrafast structural changes in myoglobin

Mix-and-extrude: high-viscosity sample injection towards time-resolved protein crystallography

FLEXR: automated multi-conformer model building using electron-density map sampling

Contact Info

Product

Resources

About