Journal of Applied Crystallography

Monte Carlo neutron ray-tracing simulations of time-of-flight (TOF)-Laue neutron macromolecular crystal diffraction (n-MX) using the McStas software package were done for the upcoming NMX Macromolecular Diffractometer at the European Spallation Source. Splitting neutron rays that arrive at the crystal lead to dramatic improvements in event formation with minimal computational overhead. The simulated event probability data was sampled using a new single-pass weighted reservoir sampling method, and processed like real n-MX data using DIALS. The effects of air and beamstop scatter on simulated data was investigated. SynopsisMonte Carlo simulations of neutron protein diffraction experiments provide useful data that models instrumental components that interact with neutrons, as well as the crystal diffraction itself. These data can be applied to instrument development, such as the commissioning of the NMX Macromolecular Diffractometer at ESS.

Capturing meta-stable conformations of enzymes and ligand complexes demands structural snapshots beyond static crystal structures. While time-resolved serial crystallography at room temperature, offers a time-resolution down to the femto-second domain it requires large amounts of micro crystals, specialized beamlines and considerable experience. Moreover, as the majority of enzymes displays turnover-times in the millisecond domain or slower, simpler methods can provide meaningful structural insight into enzyme catalysis. Vitrification of protein crystals can trap reaction intermediates by rapid cooling to {inverted exclamation} 100 K, and has traditionally been used to gain insight into long lived reaction intermediates such as product complexes. However, manual vitrification procedures are limited to long delay times of at least several seconds and heavily suffer from operator variability. A solution to this problem is provided by automatic crystal plunging devices, such as the Spitrobot, that plunge loop-mounted protein crystals into liquid nitrogen within millisecond time-scales. Versatile means of reaction initiation can be achieved either by micro dispensing a ligand droplet, or via optical excitation of light-sensitive proteins, or via the photoactivation of caged compounds. In addition to the conceptual simplicity, another benefit of cryo-trapping is that data can be collected at conventional synchrotron beamlines, exploiting their robust high-throughput capabilities. Thus, compared to room-temperature time-resolved crystallography, users not only benefit from uncoupling sample-preparation and data-collection, but also from a reduction in the required technical expertise and ready access to radiation sources. However, as cryo-trapping crystallography explores dynamic structural changes that become only visible by the comparison of several samples, experiments have to be carefully planned to carry out the necessary controls and to avoid mis- or over-interpretation of the results. Here we describe a detailed protocol for cryo-trapping time-resolved crystallography using automated crystal-plungers that enables researchers to map enzymatic reaction coordinate pathways within the millisecond domain.

Ligand binding has been shown to induce significant alterations in the conformational landscape of proteins. Traditional crystallography approaches have provided valuable input about the end states in ligand-binding reactions. However, dynamical relationships between ligand binding and backbone rearrangement often remain obscured by crystallographic structures. In the present study, we use time-resolved serial synchrotron crystallography (TR-SSX) to directly visualize indole binding in the cavity of T4 lysozyme L99A in microcrystals under controlled environmental conditions. By integrating fixed target crystallography with LAMA-based ligand delivery, we have been able to track the progression of ligand binding and backbone rearrangement. By utilizing an occupancy refinement protocol, we have been able to quantify structural populations. Our studies reveal that ligand binding for this protein cavity follows a diffusion-limited process that progressively rearranges the F -helix of the protein towards a dominant conformational state. These findings establish an observable link between ligand diffusion, occupancy evolution and conformational adaptation within a crystalline environment. More broadly, our work shows how TR-SSX can quantify ligand and conformational populations during binding, providing a framework to interpret structural adaptation in real time.

Calculation of density maps from atomic models is essential for structural studies using crystallography and electron cryo-microscopy (cryoEM). These maps serve various purposes, including atomic model building, refinement, visualization, and validation. However, accurately comparing model-calculated maps to experimental data poses challenges, particularly because the resolution of cryoEM experimental maps varies across the map. Traditional crystallography methods generate finite-resolution maps with uniform resolution throughout the unit cell volume, while most modern software in cryoEM employ Gaussian-like functions to generate these maps, which does not adequately account for atomic model parameters and resolution. Recent work by Urzhumtsev & Lunin (2022, IUCr Journal, 9, 728-734) introduces a novel method for computing atomic model maps that incorporate local resolution and can be expressed as analytically differentiable functions of all atomic parameters. This approach enhances the accuracy of matching atomic models to experimental maps. In this paper, we detail the implementation of this method in CCTBX and Phenix. SynopsisNew tools implemented in CCTBX and Phenix allow the calculation of variable-resolution maps through a sum of atomic images expressed as analytic functions of all atomic parameters, along with their associated local resolution.

Cryo-Focused Ion Beam Scanning Electron Microscopy (cryoFIB-SEM) using samples fixed by high-pressure freezing uniquely enables high resolution cryo-volume Electron Microscope (cvEM) images of cell ultrastructure to be obtained from whole cells and complex tissues in their near native state. As the freezing process also preserves fluorescence, the link between three-dimensional (3D) ultrastructure and biological process is also enabled by targeted cryo-Correlative Light and Electron Microscopy (CLEM). However, the overall viability of cvEM is challenged by sample preparation, charge balance during imaging, sample sensitivity to beam damage, contamination, and very long acquisition times. Here we detail new experimental workflows to significantly reduce each of these effects and demonstrate the improvement in resolution possible with results from the nematode Caenorhabditis elegans and the ciliated protozoon Paramecium bursaria containing many endosymbiotic algae. These results demonstrate the versatility and potential wide-ranging utility of cvEM for 3D ultrastructural imaging of whole multicellular and unicellular organisms.

Efficient protein synthesis in eukaryotic cells typically requires a 5' cap structure on messenger RNAs (mRNAs). However, under stress conditions or in viral infection, translation can also occur independently of the cap via internal ribosomal entry sites (IRES). IRES elements are therefore key regulators of protein expression in both viral and cellular contexts. Here we describe a cell-free protocol to quantitatively assess IRES-mediated translation using wheat germ extract (WGE) and a firefly luciferase (FLuc) reporter. The protocol includes template preparation, RNA synthesis and luminescence measurement following in vitro translation in WGE. This method enables rapid and robust comparison of IRES activity under controlled conditions and can additionally be applied to evaluate mRNA modifications designed to enhance translation efficiency. Key featuresO_LIStringent in vitro workflow from DNA template preparation through RNA synthesis and protein synthesis to reporter readout, including quality controls. C_LIO_LIEvaluation of IRES-driven translation suitable for testing combinations of IRES and CDS. C_LIO_LItranslation analysis without radioactive labeling. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/716985v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@417649org.highwire.dtl.DTLVardef@1bcd186org.highwire.dtl.DTLVardef@15fecb3org.highwire.dtl.DTLVardef@acdf8d_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical AbstractPipeline for the production and evaluation of IRES-firefly luciferase constructs using wheat germ extract. (1-4) Preparation: IRES-firefly luciferase constructs are amplified in E. coli and isolated from bacterial cells. Plasmids are linearized to prepare for in vitro transcription. (5-6) Transcript synthesis and verification: In vitro transcription is followed by electrophoretic validation to confirm integrity and correct molecular weight. (7-8) Translation and detection: Translation is executed in wheat germ extract and quantified by measuring reporter activity in a luminometer.

The rational design of biomolecule immobilization strategies requires molecular-level understanding of how surface properties, tethering geometry, and structural dynamics jointly influence stability and function. Recently, coarse-grained molecular dynamics simulations based on the Martini force field have emerged as an efficient framework for studying enzyme-surface interactions. However, the reproducible construction of immobilized systems with controlled orientations remains technically challenging, usually involving multiple computational tools. Here we present MartiniSurf, an open-source command-line framework for the preparation of protein and DNA systems immobilized on solid supports within the Martini paradigm. MartiniSurf integrates automated structure retrieval and cleaning, coarse graining via tools from the Martini force field software ecosystem, customizable surface generation, and biomolecule orientation based on user-defined anchoring residues, producing complete GROMACS-ready simulation systems. The framework supports both implicit restraint-based anchoring and explicit linker-mediated immobilization, including surfaces functionalized with user-defined ligands or linker-like moieties, enabling representation of mono- and multivalent attachment geometries at different modeling resolutions. Structure-based G[o]Martini potentials can be incorporated for proteins, while DNA systems are modeled using Martini 2. Optional substrate insertion, pre-coarse-grained complex handling, and automated solvation and ionization further extend system flexibility. By integrating these components into a unified workflow, MartiniSurf enables systematic and high-throughput in silico exploration of surface-tethered biomolecules and provides a robust computational platform for rational immobilization studies. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/714767v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@bc1ac4org.highwire.dtl.DTLVardef@1813b43org.highwire.dtl.DTLVardef@159b19borg.highwire.dtl.DTLVardef@19b60d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionTrabecular bone exhibits brittle behaviour governed by microscale deformation and damage processes, yet quantitative characterisation of crack progression remains challenging because classical fracture mechanics approaches do not apply to architecturally discontinuous porous tissues. This study evaluates whether synchrotron X-ray computed tomography (XCT) combined with digital volume correlation (DVC) can provide a practical experimental approach for quantifying crack opening behaviour in human trabecular bone. MethodSemicylindrical specimens harvested from femoral heads of hip-fracture donors (n = 5) and non-fracture controls (n = 5) underwent stepwise three-point-bending during XCT imaging. Full-field displacement maps enabled direct measurement of crack mouth opening displacement (CMOD), crack length (a), and their ratio, CMOD/a, used here as a geometry-normalised comparative descriptor of brittle response. Automated crack segmentation using phase-congruency crack detection (PCCD) was compared against manual measurements. ResultsXCT-DVC successfully resolved three-dimensional displacement discontinuities during crack initiation and propagation in all specimens. Hip-fracture donors exhibited significantly lower critical crack-opening ratios (CMOD/a)* than Controls (0.31 vs 0.47; p = 0.008) and reached mechanical instability at lower applied loads, consistent with a more brittle structural response under this test configuration. Despite these differences, total crack extension ({Delta}a*) was similar between groups. Automated crack tracking using phase-congruency-based segmentation showed excellent agreement with manual measurements (r{superscript 2} = 0.98), confirming reliable extraction of crack geometry from DVC displacement fields. DiscussionThese results indicate that XCT-DVC can provide a practical approach for quantifying crack-opening behaviour in trabecular bone when classical fracture-mechanics parameters are not applicable in anatomically constrained specimens. The reduced critical crack-opening ratios and earlier instability observed in Hip-fracture donors are consistent with a more brittle comparative mechanical response that is not captured by crack extension alone. The strong agreement between automated and manual crack measurements further supports displacement-based descriptors as reliable comparative indicators of brittle behaviour in porous, architecturally discontinuous tissues. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/714043v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@31c5d7org.highwire.dtl.DTLVardef@1b3d9a4org.highwire.dtl.DTLVardef@95df7borg.highwire.dtl.DTLVardef@1834216_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

Fluorescent reporters such as fluorescent proteins or chemigenetic indicators are indispensable tools for studying biological processes using light microscopy. Choosing an appropriate fluorescent tag is a crucial step in experimental design not only for imaging but also for quantitative measurements such as fluorescence fluctuation spectroscopy. Two key parameters should be considered: Fluorescent brightness and photo-bleaching. Change to fluorescence intensity due to photobleaching is relatively easy to assess in different biological environments, while brightness is more elusive. Here, we develop and employ a fluorescence correlation spectroscopy (FCS) based excitation scan assay that determines fluorescent protein performance and validate it in tissue culture and zebrafish embryos. We employ our FCS pipeline to compare a set of 10 established fluorescent proteins as well as HALO and SNAP tags for both cellular imaging and measurements of diffusion dynamics with FCS. We show that mNeonGreen outperforms mEGFP in tissue culture and zebrafish embryos. We also compare StayGold variants against other green fluorescent proteins and chemigenetic reporters in tissue culture. Overall, we present a broadly applicable approach for determining fluorescent reporter brightness in the living system of interest.

We present mdBIRCH, an online clustering method that adapts the BIRCH CF-tree to molecular dynamics (MD) data by using a merge test calibrated directly to RMSD. Each arriving frame is routed to the nearest centroid and added only if the post-merge radius computed from the cluster feature remains within a user-supplied threshold. This keeps the average deviation to each cluster centroid bounded as the cluster grows and preserves a simple interpretation of resolution in physical units. We evaluate mdBIRCH on a {beta}-heptapeptide and the HP35 system. We propose two protocols to make the threshold selection easier: (a) RMSD-anchored runs that use controlled structural edits to define interpretable operating points and (b) blind sweep that tracks how cluster count, occupancy, and coverage change with the threshold. In both systems, increasing the threshold reduces the number of clusters, concentrates coverage in high-occupancy states, and broadens within-cluster RMSD distributions. Furthermore, because decisions rely only on cluster summaries, mdBIRCH completely avoids the need for pairwise distance matrices, scales near-linearly with the number of frames on standard hardware, and naturally supports incremental operation. The method offers a practical combination of speed and interpretability for large-scale trajectory analysis.

Bacteria microcompartments (BMCs) are pseudo-organelles comprised of a self-assembling, semi-permeable protein shell, most commonly enclosing components of enzymatic pathways. -Carboxysomes (-CBs) are anabolic BMCs known for their role in sequestering Rubisco, the enzyme responsible for carbon fixation in plants, algae and bacteria, along with an upstream enzyme and an assembly protein. Rubisco has low selectivity for its substrate, CO2, and has a slow enzymatic turnover rate, resulting in an inefficient metabolic pathway. Within the -CB, Rubisco has been observed at a range of concentrations and with either a liquid-like assembly or a pseudo-lattice of polymerized fibrils. The biophysical origins of the fibril ultrastructure organization are unclear; however, it is only observed inside -CBs. Quantitative knowledge of the binding constants and energies for assembly and maintenance of these fibrils is critical for understanding this organization and Rubisco regulation, but quantitative methods for in situ analysis of Rubisco polymerization have been lacking. Here, we present an approach to convert tomography-derived -CB volumes and Rubisco particle positions into polymerization binding curves. We used this procedure to determine the Rubisco polymerization constants, including the nucleus size (n) and equilibrium polymerization constant (Kpol). The adopted modeling approach is consistent with in situ constraints, such as concentration-dependent binding interactions and confinement. This approach offers a powerful tool to evaluate both in vitro and potentially in vivo biomolecular interactions, both of Rubisco and of other proteins and polymers suitable for analysis by cryo-electron tomography. Significance StatementCryogenic electron tomography (cryoET) is a powerful method to resolve structures of proteins in their native environment at subnanometer-level resolution. Because tomography data retains spatial relationships of all particles, it intrinsically contains information about component (e.g., protein) binding interactions. Here, we use Rubisco polymerization in -carboxysomes as a model system to demonstrate that quantitative, biochemical binding analysis is possible with cryoET.

Although machine learning has transformed protein structure prediction of folded protein ground states with remarkable accuracy, intrinsically disordered proteins and regions (IDPs/IDRs) are defined by diverse and dynamical structural ensembles that are predicted with low confidence by algorithms such as AlphaFold and RoseTTAFold. We present a new machine learning method, IDPForge (Intrinsically Disordered Protein, FOlded and disordered Region GEnerator), that exploits a transformer protein language diffusion model to create all-atom IDP ensembles and IDR disordered ensembles that maintains the folded domains. IDPForge does not require sequence-specific training, back transformations from coarse-grained representations, nor ensemble reweighting, as in general the created IDP/IDR conformational ensembles show good agreement with solution experimental data, and options for biasing with experimental restraints are provided if desired. We envision that IDPForge with these diverse capabilities will facilitate integrative and structural studies for proteins that contain intrinsic disorder, and is available as an open source resource for general use.

Digital holographic microscopy systems in a common-path configuration, compared to systems with a separate reference arm, offer a compact design and resistance to disturbances. They can operate with partially coherent illumination, reducing speckle noise. However, they are limited by the overlapping of the object beam and its laterally shifted replica. As a result, images from different regions of the object overlap on the detector, preventing imaging of dense samples. We present the wavelength-scanning replica-removal method, which solves this problem by enabling the separation of information from both replicas and thereby doubling the effective field of view (FOV). The wavelength-scanning multi-shear replica removal algorithm plays a key role in reconstructing the undisturbed phase from a series of holograms recorded with variable shears. The shear value is controlled by changing the illumination wavelength. This enabled the development of two measurement modes: time-domain wavelength scanning for high-quality imaging, and a single-shot mode with frame division into color channels to improve temporal resolution. The method was validated using resolution tests and biological samples - neurons and dynamic yeast cultures. By combining the advantages of the common-path configuration with dense-structure imaging and dynamic processes, the proposed method constitutes a versatile tool for quantitative phase microscopy.

Affinity chromatography is a powerful and therefore popular method for the purification of proteins for structural studies. The success of the technique relies on the specificity of the interaction between the target protein and the affinity resin. Here, we present the identification of two protein contaminants isolated from HEK293 cell lysate following affinity purification of twin Strep-tagged or FLAG-tagged proteins. The contaminants were identified as human propionyl-coenzyme A carboxylase (hPCC) and protein arginine methyltransferase 5 in complex with methylosome protein 50 (PRMT5:MEP50) via a combination of cryo-EM data processing and proteomic analyses. This report serves to illustrate how these contaminants may appear in cryo-EM datasets and to highlight the paramount importance of affinity chromatography resin specificity for efficient protein purification.

Coccolithophores are unicellular marine phytoplankton that produce complex and intricately shaped mineralised scales called coccoliths. Coccoliths are produced in an intracellular vesicle where crystal nucleation occurs, from which several individual calcite units develop with anisotropic crystallographic facets, prompting studies into the cellular mechanisms which control crystal growth within the cell. Here, we characterise those morphological developments in 3D that occur during the formation of coccoliths by the species Gephyrocapsa huxleyi using cryo-ptychographic X-ray computed tomography. This technique is ideally suited to study coccolith mineral development, as intracellular structures can be imaged intact in their native state without needing to disrupt cells. Combined with additional imaging of developing coccoliths using cryo-transmission electron microscopy and scanning electron microscopy, we report the developmental stages involved in coccolith growth across the complete mineralisation period, while also showing that the constrained space created by individual crystal units growing in close confinement affects the final crystal morphology and overall mineral structure. These findings provide clarification on the mineralisation pathways that coccolithophores and other biomineralising organisms use to control the formation of highly functionalised crystalline structures, particularly relevant in the design of materials with tunable properties.

Single-molecule localization microscopy (SMLM) methods enable fluorescence imaging of biological specimens with nanometer-scale resolution. Although fluorophore localization precision is theoretically limited only by photon statistics, in practice the resolution of SMLM images is often degraded by physical drift of the sample and/or the microscope during data acquisition. At present, correcting this effect requires either specialized stabilization systems or computationally intensive post-processing, and established drift correction algorithms based on image cross-correlation suffer from limited temporal resolution. In this study we introduce COMET, a new method for SMLM drift estimation which achieves a substantially higher precision, accuracy, and temporal resolution compared with existing algorithmic approaches. We demonstrate that improved drift estimation translates directly into higher SMLM image resolution, limited by localization precision rather than drift artifacts. COMET is applicable to all types of SMLM data, operating directly on 2D or 3D localization datasets, and is readily integrated into analysis workflows. We benchmark its performance using both simulations and experiments, including STORM, MINFLUX, and Sequential OligoSTORM measurements, where long acquisition times make drift correction particularly challenging. COMET is published as an open-source, Python-based software project and is also available on open cloud-computing platforms.

Accurate ion force fields are essential for molecular dynamics simulations of biomolecular systems, particularly in combination with modern water models such as OPC. While OPC water improves the description of bulk water and biomolecules, the transferability of existing ion force fields to this model remains an open question. Here, we systematically assess the transferability of monovalent and divalent ion force field parameters (Li+, Na+, K+, Cs+, Mg2+,Ca2+, Sr2+, Ba2+, Cl- and Br-) to OPC water by comparing single-ion and ion-pairing properties with experimental data. Our analysis reveals that no single literature parameter set provides accurate results for all ions when directly transferred to OPC water. We hence introduce the MS/G-LB(OPC) force field, which combines Mamatkulov-Schwierz-Grotz cation parameters with Loche-Bonthuis anion parameters. MS/G-LB(OPC) reproduces hydration free energies, first-shell structural properties and activity derivatives at low salt concentrations. Our results demonstrate that transferring ion parameters to OPC can lead to significant and ion-specific deviations from experimental data, making careful validation essential. At the same time, the systematic transfer and combination of ion parameters from existing force fields can provide a practical and computationally efficient alternative to full reparameterization. MS/G-LB(OPC) is available at https://git.rz.uni-augsburg.de/cbio-gitpub/opc-ion-force-fields.

Human RNase 2 (eosinophil-derived neurotoxin, EDN) is a major eosinophil granule protein of the vertebrate-specific RNase A superfamily and is involved in antiviral response and inflammation. Identifying ligand-binding pockets in EDN is thus relevant to structure-based drug design. In our laboratory we identified by protein crystallography a conserved site at the protein surface binding to carboxylic anion molecules (malonate, tartrate and citrate). Searching for potential biomolecules rich in anion groups and considering previous report of EDN binding to glycosaminoglycans, we explored the protein binding to saccharides. Next, EDN crystals were soaked with mono- and disaccharides, and the 3D structures of ten complexes were solved by X-ray crystallography at atomic resolution. We identified protein binding pockets to glucose, fucose, mannose, sucrose, galactose, trehalose, N-acetyl-D-glucosamine, N-acetylmuramic acid, and the sialic acid N-acetylneuraminic acid. A main site for glucose, fucose, and galactose was located adjacent to the spotted carboxylic anion site. Secondarily, N-acetylneuraminic acid, N-acetylmuramic acid, sucrose, galactose, and mannose shared another protein surface region. Overall, the saccharides clustered into seven defined sites, outlining a conserved recognition pattern, which was further analysed by molecular modelling. Interestingly, within the RNase A family, we find amphibian RNases that were initially isolated as carbohydrate binding proteins and named as leczymes, combining enzymatic and lectin properties. The present data is the first systematic structural characterization of a mammalian sugar-binding RNase within the family. The results highlight unique EDN residues that mediate its sugar specific interactions, of particular interest for a better understanding of the protein physiological role. HighlightsO_LIstructure of RNase 2 in complex with mono and disaccharides at atomic resolution C_LIO_LIidentification of RNase 2 unique sugar binding sites C_LIO_LIcharacterization of a mammalian RNase A family enzyme with lectin properties C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/713198v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1d805f7org.highwire.dtl.DTLVardef@16fcc49org.highwire.dtl.DTLVardef@ccfd92org.highwire.dtl.DTLVardef@1b8f1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

The core biopolymers (DNA, RNA and proteins) are assembled from their monomers under conditions that avoid water. RNA is crucial for the Origin of Life. When cleaved from its polymerized state, RNA first transitions to nucleoside 2,3-cyclic phosphates. In the reverse direction, RNA polymerizes from 2,3-cyclic monomers in dry states, creating short oligomers that then can ligate on a template under aqueous, alkaline conditions. We studied the role of the counterions in polymerization of 2,3-cyclic nucleotides under geologically plausible settings. Through experiments and simulations, we demonstrate that the presence of ammonium and alkylammonium counterions greatly improves RNA polymerization. The otherwise less reactive cytidine containing monomers formed polyC sequences of up to heptamers; copolymers of AU, GC, or GCAU were detected up to hexamers. Our findings suggest three reasons for this: (1) (Alkyl)ammonium cations form hydrogen bonds with phosphates, (2) their alkaline pKa value can trigger general base catalysis, and (3) (alkyl)ammonium salts naturally form dry, anhydrous materials. The findings indicate that pyrolyzed organic tars creating ammonia-rich gas pockets in subsurface rocks could have enhanced the early evolution of RNA. TOC image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/713775v1_ufig1.gif" ALT="Figure 1"> View larger version (112K): org.highwire.dtl.DTLVardef@1adc431org.highwire.dtl.DTLVardef@12b8da0org.highwire.dtl.DTLVardef@5f187dorg.highwire.dtl.DTLVardef@140ed1a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Purified proteins are routinely flash frozen for use in functional and structural studies, providing a convenient way to reproduce results across complex experiments. Rho guanine-nucleotide exchange factors (RhoGEFs) are no exception to this practice, yet the effects of freezing on their activity and stability remain largely uncharacterized. This gap potentially affects the characterization of these important enzymes and how results are interpreted with respect to their prospective use as therapeutic targets. Here, we tested the isolated DH/PH tandems of P-Rex1, P-Rex2, and PRG under different cryoprotectant conditions and monitored activity and thermostability over time after flash freezing. Our results show a clear divergence between the activity of fresh and frozen purified RhoGEF protein samples in as little as one week for some conditions. Specifically, the variability in data collected on frozen samples was greatly increased. Despite these differences, thermostability seems to be preserved for much longer timepoints across RhoGEFs. Moreover, despite eventual changes in both activity and thermostability with respect to freezing, there are no obvious changes in global conformation between fresh and frozen samples of the isolated P-Rex2 DH/PH tandem. From our data, there are few generalizable trends between the different RhoGEFs and no single cryoprotective agent tested was a silver bullet to preserve both activity and thermostability across RhoGEFs. Overall, our findings emphasize the unpredictable effects of freezing RhoGEFs. As such, RhoGEF freezing should be carefully characterized for each protein and critically viewed when comparing analyses between different studies.