Spatial distribution of radiation damage to crystalline proteins at 25–300 K

Warkentin, Matthew; Badeau, Ryan; Hopkins, Jesse B.; Thorne, Robert

doi:10.1107/s0907444912021361

Cited by 20 publications

(19 citation statements)

References 69 publications

(95 reference statements)

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“…Complementary to limiting radiation damage during data collection, it is prudent to check for its manifestations before any structural analysis that might lead to inferences about biology. The effects of damage are mostly global, but partially local: for example at solvent-exposed instead of buried regions (Warkentin et al, 2012). Other specific local effects include the decarboxylation of Asp and Glu side chains and the breakage of disulfide bonds.…”

Section: Data-collection Improvementsmentioning

confidence: 99%

Journey to the center of the protein: allostery from multitemperature multiconformer X-ray crystallography

Keedy

2019

Acta Crystallogr D Struct Biol

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Proteins inherently fluctuate between conformations to perform functions in the cell. For example, they sample product-binding, transition-state-stabilizing and product-release states during catalysis, and they integrate signals from remote regions of the structure for allosteric regulation. However, there is a lack of understanding of how these dynamic processes occur at the basic atomic level. This gap can be at least partially addressed by combining variable-temperature (instead of traditional cryogenic temperature) X-ray crystallography with algorithms for modeling alternative conformations based on electron-density maps, in an approach called multitemperature multiconformer X-ray crystallography (MMX). Here, the use of MMX to reveal alternative conformations at different sites in a protein structure and to estimate the degree of energetic coupling between them is discussed. These insights can suggest testable hypotheses about allosteric mechanisms. Temperature is an easily manipulated experimental parameter, so the MMX approach is widely applicable to any protein that yields well diffracting crystals. Moreover, the general principles of MMX are extensible to other perturbations such as pH, pressure, ligand concentration etc. Future work will explore strategies for leveraging X-ray data across such perturbation series to more quantitatively measure how different parts of a protein structure are coupled to each other, and the consequences thereof for allostery and other aspects of protein function.

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Section: Data-collection Improvementsmentioning

confidence: 99%

Journey to the center of the protein: allostery from multitemperature multiconformer X-ray crystallography

Keedy

2019

Acta Crystallogr D Struct Biol

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“…Third, crystallographic order parameters (S 2 ) weight these harmonic and non-harmonic contributions 101 in a single metric that quantifies the disorder of each residue in a multiconformer model, allowing direct 102 comparison with NMR-determined order parameters (Fenwick et al, 2014). Finally, methodological advances 103 based on the physics of ice formation have enabled variable-temperature crystallographic data collection at 104 temperatures between 300 K and 100 K with modest or no use of potentially conformation-perturbing 105 cryoprotectants (Warkentin et al, 2012;Warkentin and Thorne, 2009). Together, these methods overcome many 106 of the limitations of previous X-ray-based approaches, and will contribute to an integrated view of how protein 107 conformational heterogeneity and dynamics evolve with temperature.…”

Section: Introductionmentioning

confidence: 99%

Mapping the Conformational Landscape of a Dynamic Enzyme by Multitemperature and XFEL Crystallography

Keedy¹,

Kenner²,

Warkentin

et al. 2015

Preprint

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33Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for 34 structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) 35has been previously linked to its catalytic function, but the extent to which the different conformations of these 36 residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature 37 synchrotron crystallography and fixed-target X-ray free electron laser (XFEL) crystallography. The "diffraction-38 before-destruction" nature of XFEL experiments provides a radiation-damage-free view of the functionally 39 important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the 40 temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. 41Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and 42above, yet others remain populated or become populated at 180 K and below. These results point to a complex 43 evolution of conformational heterogeneity between 180-240 K that involves both thermal deactivation and 44 solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to 45 temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in 46 which exchange between rotamer states for a large aromatic ring in the middle of the network shifts the 47 conformational ensemble for the other residues in the network. Together, our multitemperature analyses and 48XFEL data motivate a new generation of temperature-and time-resolved experiments to structurally characterize 49 the dynamic underpinnings of protein function. 50 51 Current structural biology methods provide only incomplete pictures of how proteins interconvert between 52

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“…Data collection and refinement statistics of SaR2lox. References (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)…”

Section: Supplementary Materialsmentioning

confidence: 99%

Solving a new R2lox protein structure by microcrystal electron diffraction

Lebrette

Clabbers

et al. 2019

Sci. Adv.

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Microcrystal electron diffraction (MicroED) has recently shown potential for structural biology. It enables the study of biomolecules from micrometer-sized 3D crystals that are too small to be studied by conventional x-ray crystallography. However, to date, MicroED has only been applied to redetermine protein structures that had already been solved previously by x-ray diffraction. Here, we present the first new protein structure—an R2lox enzyme—solved using MicroED. The structure was phased by molecular replacement using a search model of 35% sequence identity. The resulting electrostatic scattering potential map at 3.0-Å resolution was of sufficient quality to allow accurate model building and refinement. The dinuclear metal cofactor could be located in the map and was modeled as a heterodinuclear Mn/Fe center based on previous studies. Our results demonstrate that MicroED has the potential to become a widely applicable tool for revealing novel insights into protein structure and function.

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Spatial distribution of radiation damage to crystalline proteins at 25–300 K

Cited by 20 publications

References 69 publications

Journey to the center of the protein: allostery from multitemperature multiconformer X-ray crystallography

Journey to the center of the protein: allostery from multitemperature multiconformer X-ray crystallography

Mapping the Conformational Landscape of a Dynamic Enzyme by Multitemperature and XFEL Crystallography

Solving a new R2lox protein structure by microcrystal electron diffraction

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