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Mutants of the C 4 -symmetric protein RhuA were designed to self-assemble into two-dimensional crystalline lattices with precise spatial arrangements and patterns; the lattices of one of the variants are auxetic and deform perpendicularly to an applied force in a way that is contrary to what is generally expected in typical materials. Protein assemblies designed to surprise Auxetic materials are those that, because of their internal structure, deform perpendicularly to an applied force in a manner opposite to what is generally expected. So, when stretched, they get thicker across their width, and when compressed they get thinner. Akif Tezcan and colleagues have created a crystalline protein lattice that demonstrates such behaviour, dependent on the positioning and type of linkages between each individual protein unit. They designed mutants of the C 4 -symmetric protein RhuA to self-assemble into two-dimensional crystalline lattices with precise spatial arrangements and patterns. Disulfide bonds and metal-mediated coordination between units provide a balance between robustness and flexibility, such that large, low-defect lattices are formed that exhibit coherent rotational motion in response to an applied stress. Two-dimensional (2D) crystalline materials possess unique structural, mechanical and electronic properties 1 , 2 that make them highly attractive in many applications 3 , 4 , 5 . Although there have been advances in preparing 2D materials that consist of one or a few atomic or molecular layers 6 , 7 , bottom-up assembly of 2D crystalline materials remains a challenge and an active area of development 8 , 9 , 10 . More challenging is the design of dynamic 2D lattices that can undergo large-scale motions without loss of crystallinity. Dynamic behaviour in porous three-dimensional (3D) crystalline solids has been exploited for stimuli-responsive functions and adaptive behaviour 11 , 12 , 13 . As in such 3D materials, integrating flexibility and adaptiveness into crystalline 2D lattices would greatly broaden the functional scope of 2D materials. Here we report the self-assembly of unsupported, 2D protein lattices with precise spatial arrangements and patterns using a readily accessible design strategy. Three single- or double-point mutants of the C 4 -symmetric protein RhuA were designed to assemble via different modes of intermolecular interactions (single-disulfide, double-disulfide and metal-coordination) into crystalline 2D arrays. Owing to the flexibility of the single-disulfide interactions, the lattices of one of the variants ( C98 RhuA) are essentially defect-free and undergo substantial, but fully correlated, changes in molecular arrangement, yielding coherently dynamic 2D molecular lattices. C98 RhuA lattices display a Poisson’s ratio of −1—the lowest thermodynamically possible value for an isotropic material—making them auxetic.

The structural dynamics of a molecule are determined by the underlying potential energy landscape. Conical intersections are funnels connecting otherwise separate potential energy surfaces. Posited almost a century ago 1 , conical intersections remain the subject of intense scientific interest 2 – 5 . In biology, they have a pivotal role in vision, photosynthesis and DNA stability 6 . Accurate theoretical methods for examining conical intersections are at present limited to small molecules. Experimental investigations are challenged by the required time resolution and sensitivity. Current structure-dynamical understanding of conical intersections is thus limited to simple molecules with around ten atoms, on timescales of about 100 fs or longer 7 . Spectroscopy can achieve better time resolutions 8 , but provides indirect structural information. Here we present few-femtosecond, atomic-resolution videos of photoactive yellow protein, a 2,000-atom protein, passing through a conical intersection. These videos, extracted from experimental data by machine learning, reveal the dynamical trajectories of de-excitation via a conical intersection, yield the key parameters of the conical intersection controlling the de-excitation process and elucidate the topography of the electronic potential energy surfaces involved. Serial femtosecond crystallography (SFX) has provided significant understanding of time-resolved processes of various systems in biology, for example, rhodopsin, which underlies our vision. The approach involves femtosecond-length X-ray pulses directed at protein crystals and has been used to study various photoactive proteins. However, the function of proteins such as rhodopsin requires trans – cis isomerization of a chromophore, which involves crossing of a conical intersection—a funnel separating potential energy surfaces—at timescales faster than what can be achieved experimentally. Here, Ourmazd and colleagues report a machine learning analysis of SFX data of photoactive yellow protein, which resolves the protein passing through a conical intersection, providing information about the potential energy surfaces involved and achieving time resolution of less than 10 fs. This approach offers an opportunity to understand some of the fastest processes in biology by extracting even more information from SFX datasets.

This article is one of ten reviews selected from the Annual Update in Intensive Care and Emergency medicine 2016. Other selected articles can be found online at http://www.biomedcentral.com/collections/annualupdate2016 . Further information about the Annual Update in Intensive Care and Emergency Medicine is available from http://www.springer.com/series/8901 .

Since preparative chromatography is a sustainability challenge due to large amounts of consumables used in downstream processing of biomolecules, protein crystallization offers a promising alternative as a purification method. While the limited crystallizability of proteins often restricts a broad application of crystallization as a purification method, advances in molecular biology, as well as computational methods are pushing the applicability towards integration in biotechnological downstream processes. However, in industrial and academic settings, monitoring protein crystallization processes non-invasively by microscopic photography and automated image evaluation remains a challenging problem. Recently, the identification of single crystal objects using deep learning has been the subject of increased attention for various model systems. However, the advancement of crystal detection using deep learning for biotechnological applications is limited: robust models obtained through supervised machine learning tasks require large-scale and high-quality data sets usually obtained in large projects through extensive manual labeling, an approach that is highly error-prone for dense systems of transparent crystals. For the first time, recent trends involving the use of synthetic data sets for supervised learning are transferred, thus generating photorealistic images of virtual protein crystals in suspension (PCS) through the use of ray tracing algorithms, accompanied by specialized data augmentations modelling experimental noise. Further, it is demonstrated that state-of-the-art models trained with the large-scale synthetic PCS data set outperform similar fine-tuned models based on the average precision metric on a validation data set, followed by experimental validation using high-resolution photomicrographs from stirred tank protein crystallization processes.

Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse. This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using 4.3 nm radiation. The operation of gas-dynamic nozzles that produce liquid microjets with velocities greater than 80 m s −1 was demonstrated. Furthermore, this article provides optical images of X-ray-induced explosions together with Bragg diffraction from protein microcrystals exposed to trains of X-ray pulses repeating at rates of up to 4.5 MHz. The results indicate the feasibility for megahertz serial crystallography measurements with hard X-rays and give guidance for the design of such experiments.

Microgravity has been shown to be an excellent tool for protein crystal formation. A retrospective analysis of all publicly available crystallization data, including many that have not yet been published, clearly demonstrates the value of the microgravity environment for producing superior protein crystals. The parameters in the database (the Butler Microgravity Protein Crystal Database, BμCDB) that were evaluated pertain to both crystal morphology and diffraction quality. Success metrics were determined as improvements in size, definition, uniformity, mosaicity, diffraction quality, resolution limits, and B factor. The proteins in the databases were evaluated by molecular weight, protein type, the number of subunits, space group, and Mattew’s Coefficient. Compared to ground experiments, crystals grown in a microgravity environment continue to show improvement across all metrics evaluated. General trends as well as numerical differences are included in the assessment of the BμCDB. The microgravity environment improves crystal formation across a spectrum of metrics and the datasets utilized for this investigation are excellent tools for this evaluation.

Background The optimal fluid management strategy for patients undergoing cardiac surgery was controversial regarding fluid volume and intraoperative fluid types. This study aimed to assess the correlation between colloids and crystalloids used for perioperative fluid therapy in cardiac surgery patients and postoperative prognosis. Methods The Ovid MEDLINE(R) ALL, Embase, and Cochrane Central Register of Controlled Trials databases were searched for eligible studies on fluid management strategies using colloids and crystalloids for cardiac surgery patients published before August 25th, 2023. Results Ten randomized controlled trials met the eligibility criteria. Compared to the use of crystalloids, the use of colloids, including hydroxyethyl starch (HES), albumin, and gelatine, did not show any differences in mortality, transfusion, acute kidney injury, and atrial fibrillation rates, postoperative blood loss, the length of hospital stay, or the length of intensive care unit (ICU) stay. The results of this meta-analysis showed that the crystalloid group had significantly reduced postoperative chest tube output compared to the colloid group. In the subgroup analysis, the amount of fresh frozen plasma (FFP) infused was significantly lower when using fluid management in the ICU and when using isotonic crystalloids compared to the colloids. In addition, when using fluid management in the ICU, patients in the colloid group had a significant increase in urine volume 24 h after surgery. However, other related factors, including the type of crystalloid solution, type of colloidal solution, and timing of liquid management, did not affect most outcomes. Conclusion Both colloids and crystalloids could be used as alternatives for perioperative fluid management after cardiac surgery. The use of crystalloids significantly reduced the postoperative chest tube output, and the need for FFP infusion decreased significantly with the use of isotonic crystalloids or fluid management during the ICU stay. ICU patients in the colloid group had higher urine output 24 h after surgery. In addition, although the infusion method was not related to most outcomes, the rates of red blood cell and FFP transfusion and postoperative blood loss in the crystalloid group seemed to be lower, which needed to be further studied in high-quality and large-sample RCTs. Trial registration PROSPERO, CRD42023415234.

High-quality crystals are essential to ensure high-resolution structural information. Protein crystals are controlled by many factors, such as pH, temperature, and the ion concentration of crystalline solutions. We previously reported the development of a device dedicated to protein crystallization. In the current study, we have further modified and improved our device. Exposure to external magnetic field leads to alignment of the crystal toward a preferred direction depending on the magnetization energy. Each material has different magnetic susceptibilities depending on the individual direction of their unit crystal cells. One of the strategies to acquire a large crystal entails controlling the nucleation rate. Furthermore, exposure of a crystal to a magnetic field may lead to new morphologies by affecting the crystal volume, shape, and quality.