Piping arrangement through a double chamber structure
Low thermal loss piping arrangement for moving cryogenic media through double chamber structure
Yager, S. P.↗
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De novo design of protein structure and function with RFdiffusion
Abstract There has been considerable recent progress in designing new proteins using deep-learning methods 1–9 . Despite this progress, a general deep-learning framework for protein design that enables solution of a wide range of design challenges, including de novo binder design and design of higher-order symmetric architectures, has yet to be described. Diffusion models 10,11 have had considerable success in image and language generative modelling but limited success when applied to protein modelling, probably due to the complexity of protein backbone geometry and sequence–structure relationships. Here we show that by fine-tuning the RoseTTAFold structure prediction network on protein structure denoising tasks, we obtain a generative model of protein backbones that achieves outstanding performance on unconditional and topology-constrained protein monomer design, protein binder design, symmetric oligomer design, enzyme active site scaffolding and symmetric motif scaffolding for therapeutic and metal-binding protein design. We demonstrate the power and generality of the method, called RoseTTAFold diffusion (RFdiffusion), by experimentally characterizing the structures and functions of hundreds of designed symmetric assemblies, metal-binding proteins and protein binders. The accuracy of RFdiffusion is confirmed by the cryogenic electron microscopy structure of a designed binder in complex with influenza haemagglutinin that is nearly identical to the design model. In a manner analogous to networks that produce images from user-specified inputs, RFdiffusion enables the design of diverse functional proteins from simple molecular specifications.
Science & Technology - Other Topics↗
Atomistic simulations of the Escherichia coli ribosome provide selection criteria for translationally active substrates
As genetic code expansion advances beyond l-α-amino acids to backbone modifications and new polymerization chemistries, delineating what substrates the ribosome can accommodate remains a challenge. The Escherichia coli ribosome tolerates non-l-α-amino acids in vitro, but few structural insights that explain how are available, and the boundary conditions for efficient bond formation are so far unknown. Here we determine a high-resolution cryogenic electron microscopy structure of the E. coli ribosome containing α-amino acid monomers and use metadynamics simulations to define energy surface minima and understand incorporation efficiencies. Reactive monomers across diverse structural classes favour a conformational space where the aminoacyl-tRNA nucleophile is <4 Å from the peptidyl-tRNA carbonyl with a Bürgi–Dunitz angle of 76–115°. Monomers with free energy minima that fall outside this conformational space do not react efficiently. This insight should accelerate the in vivo and in vitro ribosomal synthesis of sequence-defined, non-peptide heterooligomers.
59 BASIC BIOLOGICAL SCIENCES↗
The NTF Inlet Guide Vanes Thermal Gradient Problem and Its Mitigation
The National Transonic Facility (NTF) utilizes Inlet Guide Vanes (IGV) to provide precise, quick response Mach number control for the tunnel. During cryogenic operations, the massive IGV structure can experience large thermal gradients, measured as "Delta T or (Delta)T", between the IGV ring and its support structure called the transfer case. If these temperature gradients are too large, the IGV structure can be stressed beyond its safety limit and cease operation. In recent years, (Delta)T readings exceeding the prescribed safety limits were observed frequently during cryogenic operations, particularly during model access. The tactical operation methods of the tunnel to minimize (Delta)T did not always succeed. One obvious option to remedy this condition is to warm up the IGV structure by disabling the main drive operation, but this "natural" warm up method can takes days in some cases, resulting in productivity loss. This paper documents the thermal gradient problem associated with the IGV structure during cryogenic operation and how the facility has recently achieved an acceptable mitigation which has resulted in improved efficiency of operations.
Venkat, Venki S.↗
Structural Complexities in Sodium Ion Conductive Antiperovskite Revealed by Cryogenic Transmission Electron Microscopy
Here we use low-dose cryogenic transmission electron microscopy (cryo-TEM) to investigate the atomic-scale structure of antiperovskite Na 2 NH 2 BH 4 crystals by preserving the room-temperature cubic phase and carefully monitoring the electron dose. Via quantitative analysis of electron beam damage using selected area electron diffraction, we find cryogenic imaging provides 6-fold improvement in beam stability for this solid electrolyte. Cryo-TEM images obtained from flat crystals revealed the presence of a new, long-range-ordered supercell with a cubic phase. The supercell exhibits doubled unit cell dimensions of 9.4 Å × 9.4 Å as compared to the cubic lattice structure revealed by X-ray crystallography of 4.7 Å × 4.7 Å. The comparison between the experimental image and simulated potential map indicates the origin of the supercell is a vacancy ordering of sodium atoms. This work demonstrates the potential of using cryo-TEM imaging to study the atomic-scale structure of air- and electron-beam-sensitive antiperovskite-type solid electrolytes.
36 MATERIALS SCIENCE↗
Development of Structural and Thermal Elements for Critical Dynamics in Microgravity Experiment
We report on the design, fabrication and test of cryogenic sample cells and structures for the Critical Dynamics in Microgravity Experiment (DYNAMX), an experiment scheduled to fly on the space shuttle as part of the Microgravity Science Payload (MSP) in February 2001.
thermal↗
Optical Design of the Developmental Cryogenic Active Telescope Testbed (DCATT)
In the summer of 1996, three Study teams developed conceptual designs and mission architectures for the Next Generation Space Telescope (NGST). Each group highlighted areas of technology development that need to be further advanced to meet the goals of the NGST mission. The most important areas for future study included: deployable structures, lightweight optics, cryogenic optics and mechanisms, passive cooling, and on-orbit closed loop wavefront sensing and control. NASA and industry are currently planning to develop a series of ground testbeds and validation flights to demonstrate many of these technologies. The Deployed Cryogenic Active Telescope Testbed (DCATT) is a system level testbed to be developed at Goddard Space Flight Center in three phases over an extended period of time. This testbed will combine an actively controlled telescope with the hardware and software elements of a closed loop wavefront sensing and control system to achieve diffraction limited imaging at 2 microns. We will present an overview of the system level requirements, a discussion of the optical design, and results of performance analyses for the Phase 1 ambient concept for DCATT,
Davila, Pam↗
Building molecular model series from heterogeneous CryoEM structures using Gaussian mixture models and deep neural networks
Cryogenic electron microscopy (CryoEM) produces structures of macromolecules at near-atomic resolution. However, building molecular models with good stereochemical geometry from those structures can be challenging and time-consuming, especially when many structures are obtained from datasets with conformational heterogeneity. Here we present a model refinement protocol that automatically generates series of molecular models from CryoEM datasets, which describe the dynamics of the macromolecular system and have near-perfect geometry scores. This method makes it easier to interpret the movement of the protein complex from heterogeneity analysis and to compare the structural dynamics observed from CryoEM data with results from other experimental and simulation techniques.
59 BASIC BIOLOGICAL SCIENCES↗
Cryogenically formed prestressed composite fiber-metal structures for O2/N2 high pressure gas tanks.
Demonstration of high-structural-performance ARDEFORM cryoformed 301 stainless-steel glass-fiber-reinforced (GFR) vessels by room temperature tests of 13 1/2-in. diam spheres. Tests verified that the structural performance of ARDEFORM spherical GFR vessels not only exceeded that of all metal construction, but also bettered previous GFR experimental results by 50%. Achievement of essentially the full strength of fiberglass in a spherical wrap pattern was again verified. Significant weight advantages for this construction are projected for O2/N2 high-pressure gas tanks for Space Shuttle environmental control/life support system missions.
Gleich, D.↗
Characterization of Polyimide Foams for Ultra-Lightweight Space Structures
Ultra-lightweight materials have played a significant role in nearly every area of human activity ranging from magnetic tapes and artificial organs to atmospheric balloons and space inflatables. The application range of ultra-lightweight materials in past decades has expanded dramatically due to their unsurpassed efficiency in terms of low weight and high compliance properties. A new generation of ultra-lightweight materials involving advanced polymeric materials, such as TEEK (TM) polyimide foams, is beginning to emerge to produce novel performance from ultra-lightweight systems for space applications. As a result, they require that special conditions be fulfilled to ensure adequate structural performance, shape retention, and thermal stability. It is therefore important and essential to develop methodologies for predicting the complex properties of ultra-lightweight foams. To support NASA programs such as the Reusable Launch Vehicle (RLV), Clark Atlanta University, along with SORDAL, Inc., has initiated projects for commercial process development of polyimide foams for the proposed cryogenic tank integrated structure (see figure 1). Fabrication and characterization of high temperature, advanced aerospace-grade polyimide foams and filled foam sandwich composites for specified lifetimes in NASA space applications, as well as quantifying the lifetime of components, are immensely attractive goals. In order to improve the development, durability, safety, and life cycle performance of ultra-lightweight polymeric foams, test methods for the properties are constant concerns in terms of timeliness, reliability, and cost. A major challenge is to identify the mechanisms of failures (i.e., core failure, interfacial debonding, and crack development) that are reflected in the measured properties. The long-term goal of the this research is to develop the tools and capabilities necessary to successfully engineer ultra-lightweight polymeric foams. The desire is to reduce density at the material and structural levels, while at the same time maintaining or increasing mechanical and other properties.
Meador, Michael↗
Structural basis for inhibition of coagulation factor VIII reveals a shared antigenic hotspot on the C1 domain
Hemophilia A arises from dysfunctional or deficient coagulation factor (F)VIII and leads to inefficient fibrin clot formation and uncontrolled bleeding events. The development of antibody inhibitors is a clinical complication in hemophilia A patients receiving FVIII replacement therapy. LE2E9 is an anti-C1 domain inhibitor previously isolated from a mild/moderate hemophilia A patient and disrupts FVIII interactions with von Willebrand factor and FIXa, though the intermolecular contacts that underpin LE2E9-mediated FVIII neutralization are undefined. To determine the structure of the complex between FVIII and LE2E9 and characterize its mechanism of inhibition. FVIII was bound to the antigen binding fragment (Fab) of NB2E9, a recombinant construct of LE2E9, and its structure was determined by cryogenic electron microscopy. Here, this report communicates the 3.46 Å structure of FVIII bound to NB2E9, with its epitope comprising FVIII residues S2040 to Y2043, K2065 to W2070, and R2150 to H2155. Structural analysis reveals that the LE2E9 epitope overlaps with portions of the epitope for 2A9, a murine-derived inhibitor, suggesting that these residues represent a shared antigenic region on the C1 domain between FVIII –/– mice and hemophilia A patients. Furthermore, the FVIII:NB2E9 structure elucidates the orientation of the LE2E9 glycan, illustrating how the glycan sterically blocks interactions between the FVIII C1 domain and the von Willebrand factor D' domain. A putative model of the FVIIIa:FIXa complex suggests potential clashing between the NB2E9 glycan and FIXa light chain. These results describe an antigenic “hotspot” on the FVIII C1 domain and provide a structural basis for engineering FVIII replacement therapeutics with reduced antigenicity.
60 APPLIED LIFE SCIENCES↗
Mechanical response at cryogenic temperatures of selected reinforced plastic composite systems.
Structural reinforced plastic composite materials for cryogenic temperature application evaluated noting tensile, compression, shear, flexural and bearing properties
MECHANICAL PROPERTY↗
Determining biomolecular structures near room temperature using X-ray crystallography: concepts, methods and future optimization
For roughly two decades, cryocrystallography has been the overwhelmingly dominant method for determining high-resolution biomolecular structures. Competition from single-particle cryo-electron microscopy and micro-electron diffraction, increased interest in functionally relevant information that may be missing or corrupted in structures determined at cryogenic temperature, and interest in time-resolved studies of the biomolecular response to chemical and optical stimuli have driven renewed interest in data collection at room temperature and, more generally, at temperatures from the protein–solvent glass transition near 200 K to ∼350 K. Fischer has recently reviewed practical methods for room-temperature data collection and analysis [Fischer (2021), Q. Rev. Biophys. 54 , e1]. Here, the key advantages and physical principles of, and methods for, crystallographic data collection at noncryogenic temperatures and some factors relevant to interpreting the resulting data are discussed. For room-temperature data collection to realize its potential within the structural biology toolkit, streamlined and standardized methods for delivering crystals prepared in the home laboratory to the synchrotron and for automated handling and data collection, similar to those for cryocrystallography, should be implemented.
59 BASIC BIOLOGICAL SCIENCES↗
Structural concepts for future space transportation orbiters
Two structural concepts for a fully reusable STS orbiter are compared with respect to weight and the ability to satisfy structural goals. The cryogenic propellant tanks for both concepts use welded, blade-stiffened aluminum skins for leak-free containment and compatibility with LOX and LH2. The tanks are sized primarily by pressure, although some areas are sized by bending and fracture mechanics considerations. The tank concept is regarded as state-of-the-art, even though it requires a closed cell foam insulation with a dry air purge to prevent air liquefaction and ice formation. It is noted that the foam will require testing to verify its design life for use in the STS. One concept has nonintegral tanks suspended inside an insulated aluminum airframe and thrust structure. Here, a durable thermal protection system external insulation is mechanically attached to the airframe, allowing a buckled skin design. The other concept uses a novel structural arrangement consisting of a separate tank/thrust structure that supports a hot advanced-carbon-carbon aeroshell structure.
Taylor, A. H.↗
Aging Optimization of Aluminum-Lithium Alloy C458 for Application to Cryotank Structures
Compared with aluminum alloys such as 2219, which is widely used in space vehicle for cryogenic tanks and unpressurized structures, aluminum-lithium alloys possess attractive combinations of lower density and higher modulus along with comparable mechanical properties. These characteristics have resulted in the successful use of the aluminum-lithium alloy 2195 (Al-1.0 Li-4.0 Cu-0.4 Mg-0.4 Ag-0.12 Zr) for the Space Shuttle External Tank, and the consideration of newer U.S. aluminum-lithium alloys such as L277 and C458 for future space vehicles. These newer alloys generally have lithium content less than 2 wt. % and their composition and processing have been carefully tailored to increase the toughness and reduce the mechanical property anisotropy of the earlier generation alloys such 2090 and 8090. Alloy processing, particularly the aging treatment, has a significant influence on the strength-toughness combinations and their dependence on service environments for aluminum-lithium alloys. Work at NASA Marshall Space Flight Center on alloy 2195 has shown that the cryogenic toughness can be improved by employing a two-step aging process. This is accomplished by aging at a lower temperature in the first step to suppress nucleation of the strengthening precipitate at sub-grain boundaries while promoting nucleation in the interior of the grains. Second step aging at the normal aging temperature results in precipitate growth to the optimum size. A design of experiments aging study was conducted for plate. To achieve the T8 temper, Alloy C458 (Al-1.8 Li-2.7 Cu-0.3 Mg-0.08 Zr-0.3 Mn-0.6 Zn) is typically aged at 300F for 24hours. In this study, a two-step aging treatment was developed through a comprehensive 2(exp 4) full factorial design of experiments study and the typical one-step aging used as a reference. Based on the higher lithium content of C458 compared with 2195, the first step aging temperature was varied between 175F and 250F. The second step aging temperatures was varied between 275F and 325F, which is in the range of the single-step aging temperature. The results of the design of experiments used for the T8 temper as well as a smaller set of experiments for the T6 temper will be presented. The process of selecting the optimum aging treatment, based on the measured mechanical properties at room and cryogenic temperature as well as the observed deformation mechanisms, will be presented in detail. The implications for the use of alloy C458 in cryotanks will be discussed.
Sova, B. J.↗
Aging Optimization of Aluminum-Lithium Alloy C458 for Application to Cryotank Structures
Compared with aluminum alloys such as 2219, which is widely used in space vehicle for cryogenic tanks and unpressurized structures, aluminum-lithium alloys possess attractive combinations of lower density and higher modulus along with comparable mechanical properties. These characteristics have resulted in the successful use of the aluminum-lithium alloy 2195 (Al-1.0 Li-4.0 Cu-0.4 Mg-0.4 Ag-0.12 Zr) for the Space Shuttle External Tank, and the consideration of newer U.S. aluminum-lithium alloys such as L277 and C458 for future space vehicles. These newer alloys generally have lithium content less than 2 wt. % and their composition and processing have been carefully tailored to increase the toughness and reduce the mechanical property anisotropy of the earlier generation alloys such 2090 and 8090. Alloy processing, particularly the aging treatment, has a significant influence on the strength-toughness combinations and their dependence on service environments for aluminum-lithium alloys. Work at NASA Marshall Space Flight Center on alloy 2195 has shown that the cryogenic toughness can be improved by employing a two-step aging process. This is accomplished by aging at a lower temperature in the first step to suppress nucleation of the strengthening precipitate at sub-grain boundaries while promoting nucleation in the interior of the grains. Second step aging at the normal aging temperature results in precipitate growth to the optimum size. A design of experiments aging study was conducted for plate. To achieve the T8 temper, Alloy C458 (Al-1.8 Li-2.7 Cu-0.3 Mg- 0.08 Zr-0.3 Mn-0.6 Zn) is typically aged at 300 F for 24 hours. In this study, a two-step aging treatment was developed through a comprehensive 24 full factorial design of experiments study and the typical one-step aging used as a reference. Based on the higher lithium content of C458 compared with 2195, the first step aging temperature was varied between 175 F and 250 F. The second step aging temperatures was varied between 275 F and 325 F, which is in the range of the single-step aging temperature. The results of the design of experiments used for the T8 temper as well as a smaller set of experiments for the T6 temper will be presented. The process of selecting the optimum aging treatment, based on the measured mechanical properties at room and cryogenic temperature as well as the observed deformation mechanisms, will be presented in detail. The implications for the use of alloy C458 in cryotanks will be discussed.
Sova, B. J.↗
Aging Optimization of Aluminum-Lithium Alloy L277 for Application to Cryotank Structures
Compared with aluminum alloys such as 2219, which is widely used in space vehicle for cryogenic tanks and unpressurized structures, aluminum-lithium alloys possess attractive combinations of lower density and higher modulus along with comparable mechanical properties and improved damage tolerance. These characteristics have resulted in the successful use of the aluminum-lithium alloy 2195 for the Space Shuttle External Tank, and the consideration of newer U.S. aluminum-lithium alloys such as L277 and C458 for future space vehicles. A design of experiments aging study was conducted for plate and a limited study on extrusions. To achieve the T8 temper, Alloy L277 is typically aged at 290 F for 40 hours. In the study for plate, a two-step aging treatment was developed through a design of experiments study and the one step aging used as a control. Based on the earlier NASA studies on 2195, the first step aging temperature was varied between 220 F and 260 F. The second step aging temperatures was varied between 290 F and 310 F, which is in the range of the single-step aging temperature. For extrusions, two, single-step, and one two-step aging condition were evaluated. The results of the design of experiments used for the T8 temper as well as a smaller set of experiments for the T6 temper for plate and the results for extrusions will be presented.
Sova, B. J.↗
Insights into substrate coordination and glycosyl transfer of poplar cellulose synthase-8
Cellulose is an abundant cell wall component of land plants. It is synthesized from UDP-activated glucose molecules by cellulose synthase, a membrane-integrated processive glycosyltransferase. Cellulose synthase couples the elongation of the cellulose polymer with its translocation across the plasma membrane. Here, we present substrate- and product-bound cryogenic electron microscopy structures of the homotrimeric cellulose synthase isoform-8 (CesA8) from hybrid aspen (poplar). UDP-glucose binds to a conserved catalytic pocket adjacent to the entrance to a transmembrane channel. The substrate’s glucosyl unit is coordinated by conserved residues of the glycosyltransferase domain and amphipathic interface helices. Site-directed mutagenesis of a conserved gating loop capping the active site reveals its critical function for catalytic activity. Molecular dynamics simulations reveal prolonged interactions of the gating loop with the substrate molecule, particularly across its central conserved region. These transient interactions likely facilitate the proper positioning of the substrate molecule for glycosyl transfer and cellulose translocation.
59 BASIC BIOLOGICAL SCIENCES↗