Four porcine crystals, in three different unit cells, and bovine trypsin crystals, studied at 173 degrees K and 295 degrees K, were analyzed by X-ray diffraction. Crystals were grown in experiments designed to discover small molecules or additives that enhance crystallization. Mother liquors of crystals each contained a set of additives as well as known salts, buffers, and precipitants. Noteworthy were mellitic acid, pyromellitic acid, and other small organic acids and anions, benzamidine, a buffer molecule, HEPES, sucrose, and PEG fragments ranging from three to six units in length. Except for PEG molecules, most of these bound to trypsin directly and generally at protein-protein interfaces. Preponderance of carboxyl containing molecules provides further evidence of their usefulness in protein crystallization. Because of symmetry, some additive molecules were seen lying on special positions. The appearance of small molecules in protein crystals is a further testament to the value of exploring additives in crystallization screens. In porcine trypsin crystals, PEG in the solvent regions has received particular attention. Ubiquitous, extended electron density peaks plausibly interpreted as PEG molecules suggest that the solvent regions in crystals derived from PEG should be treated in a more granular manner than those using only bulk solvent corrections in modeling and refinement.

The use of polyethylene glycol in the crystallization of biological macromolecules and its appearance in the resulting crystals is discussed.

The production of enzyme microcrystals for time resolved serial crystallography employing free electron laser or synchrotron radiation is a relatively new variation on traditional macromolecular crystallization for conventional single crystal X-ray analysis. While the fundamentals of macromolecular crystal growth are the same, some modifications and special considerations are in order if the objective is to produce uniform size, microcrystals in very large numbers for serial data collection. Presented here are the basic principles of protein crystal growth with particular attention to the approaches best employed to achieve the goal of microcrystals and some novel techniques, as well as old, that may be useful. Also discussed are the advantages of particular precipitants and certain methods of growing protein crystals that might be advantageous for serial data recording.

On December 5, 2022, an indirect drive fusion implosion on the National Ignition Facility (NIF) achieved a target gain G_{target} of 1.5. This is the first laboratory demonstration of exceeding "scientific breakeven" (or G_{target}>1) where 2.05 MJ of 351 nm laser light produced 3.1 MJ of total fusion yield, a result which significantly exceeds the Lawson criterion for fusion ignition as reported in a previous NIF implosion [H. Abu-Shawareb et al. (Indirect Drive ICF Collaboration), Phys. Rev. Lett. 129, 075001 (2022)PRLTAO0031-900710.1103/PhysRevLett.129.075001]. This achievement is the culmination of more than five decades of research and gives proof that laboratory fusion, based on fundamental physics principles, is possible. This Letter reports on the target, laser, design, and experimental advancements that led to this result.

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. As recently reported, a burning plasma state, where the alpha-heating in the plasma is the primary source of heating, was achieved in laboratory experiments. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin ``burn propagation'' into surrounding cold fuel, enabling the possibility of high energy gain. While ``scientific breakeven'' (i.e. unity target gain) has not yet been achieved, this talk reports the first controlled fusion experiment on the National Ignition Facility to produce capsule gain greater than unity (here 5.8) and reach ignition by many different formulations of the Lawson criterion. In the talk, we will discuss some key basic physics inertial confinement fusion (ICF) principles behind the burning plasma and ignition results as well as discuss future challenges.

The structures of new crystal forms of Satellite tobacco mosaic virus (STMV) are described. These belong to space groups I2, P21212 (a low-resolution form), R3 (H3) and P23. The R3 crystals are 50%/50% twinned, as are two instances of the P23 crystals. The I2 and P21212 crystals were grown from ammonium sulfate solutions, as was one crystal in space group P23, while the R3 and the other P23 crystals were grown from sodium chloride, sodium bromide and sodium nitrate. The monoclinic and orthorhombic crystals have half a virus particle as the asymmetric unit, while the rhombohedral and cubic crystals have one third of a virus particle. RNA segments organized about the icosahedral twofold axes were present in crystals grown from ammonium sulfate and sodium chloride, as in the canonical I222 crystals (PDB entry 4oq8), but were not observed in crystals grown from sodium bromide and sodium nitrate. Bromide and nitrate ions generally replaced the RNA phosphates present in the I222 crystals, including the phosphates seen on fivefold axes, and were also found at threefold vertices in both the rhombohedral and cubic forms. An additional anion was also found on the fivefold axis 5 Å from the first anion, and slightly outside the capsid in crystals grown from sodium chloride, sodium bromide and sodium nitrate, suggesting that the path along the symmetry axis might be an ion channel. The electron densities for RNA strands at individual icosahedral dyads, as well as at the amino-terminal peptides of protein subunits, exhibited a diversity of orientations, in particular the residues at the ends.

The structures of Aspergillus oryzae α-amylase were determined in a tetragonal crystal, having one molecule as asymmetric unit, and a monoclinic crystal with two molecules as asymmetric unit. Both crystal forms were obtained from trace contaminants of an old commercial lipase preparation. Structures were determined and refined to 1.65 Å and 1.43 Å resolution respectively. The latter crystal has a non-crystallographic (NCS) twofold axis within the asymmetric unit. Glycosylation at Asn197 is evident, and in the tetragonal crystal can be seen to include three, partially disordered sugar residues following the initial N-acetyl glucosamine (NAG). Superposition of the tetragonal crystal model on the α-amylases from Bacillus subtilis (PDB:1BAG), pig pancreas (PDB:3L2L), and barley (PDB:1AMY), show a high degree of coincidence, particularly for the (β/α)8-barrel domains, and especially within the active site. Using this structural agreement between amylases, we extrapolated the binding model of a six residue, limit dextrin found in pig pancreas α-amylase to the A. oryzae enzyme model, which predicts substrate interacting amino acid residues.

Aim: To understand the details of the action of fungal lipase and the mechanism for its observed interfacial activation. Background: Fungal lipase, crucial to biotechnology, functions at the lipid - water interface where it undergoes a poorly understood interfacial activation. Biochemical factors influencing its activation and inhibition are also poorly understood. This study provides a basis for its activity and a plausible mechanism for interfacial activation. Objective: To determine the structures of fungal lipase in different crystal forms in complex with their enzymatic reactants and inhibitors. Method: X-ray crystallography. Results: Thermomyces lanuginosa lipase was visualized in three crystal forms, of space groups H32, P21 and I222 at 1.3 to 1.45 Å resolution. Rhombohedral crystals have one molecule, lacking segment 241 to 252, as an asymmetric unit, with molecules organized as two trimers. Monoclinic crystals’ asymmetric unit is six intact molecules organized as two, nearly identical trimers, each exhibiting an NCS threefold axis. The “lid” helix was consistently closed. Oligomerization into trimers creates an internal hydrophobic cavity where catalysis occurs. In monoclinic and orthorhombic crystals, active site serines were esterified to fatty acids. Lipase had bound within their trimeric, hydrophobic cavities 1,3-diacylglycerols with fatty acid chain lengths of about 18 carbons. Conclusions: Results suggest trimers are likely the active form of the enzyme at the lipid-water interface. Formation of trimers may provide an explanation for “interfacial activation”.

A sample of Apolipoprotein E3 used in the original structure determination by X-ray crystallography (PDB code 1NFN) was crystallized under different conditions and its structure determined by molecular replacement at 298° K. The original model (1NFN) began at amino acid 23 and ended at amino acid 164, but the amino acid segment 81 through 91 (a loop between helices) was not visible in the electron density and presumed disordered. The model reported here is essentially identical to 1NFN, but now includes amino acids 18 through 22 at the amino terminus, 165 at the carboxy terminus and includes as well the segment 83 through 91. Leu 82 is not visible, but the separation between Gln 81 and Thr 83 is more than 10 Å, thereby indicating a proteolytic cleavage occurred between those two residues.

X-ray intensities extending to 1.4 Å resolution were collected on the P63 hexagonal crystal form of canavalin, and extended to 1.9 Å for the orthorhombic C2221 crystals. Structure determination of a new crystal form of canavalin having space group P212121 is reported as well. Both the N and C terminal cupin domains contained identifiable ligands. For hexagonal crystals, in the cavity of the C terminal cupin, a molecule of benzoic acid was found, bound through carboxyl oxygens to Histidine 297, asparagine 284 and Arginine 376. The benzene ring was immersed in a cluster of at least 8 hydrophobic amino acid side chains. The N terminal cupin contained a molecule of citrate. Benzoic acid was also found to be present in the C terminal cupins of in the C2221 and P212121 crystal forms. In rhombohedral crystals, the C terminal cupin domain appeared to be occupied by a phosphate ion, but this was ambiguous. In cubic crystals, both domains were vacant. The N terminal cupin domains of canavalin in the P212121 and rhombohedral crystals were also vacant, but the N terminal cupin domain of the C2221 crystals contained a ligand whose identity is uncertain, but which has been modeled as HEPES buffer. A possible physiological role for the ligands and their complexes with canavalin is considered.

An iron-containing alcohol dehydrogenase (FeADH) from the hyperthermophilic archaeon Thermococcus thioreducens was crystallized in unit cells belonging to space groups P21, P212121 and P43212, and the crystal structures were solved at 2.4, 2.1 and 1.9 Å resolution, respectively, by molecular replacement using the FeADH from Thermotoga maritima (Schwarzenbacher et al., 2004) as a model. In the monoclinic and orthorhombic crystals the dehydrogenase (molecular mass 41.5 kDa) existed as a dimer containing a twofold noncrystallographic symmetry axis, which was crystallographic in the tetragonal crystals. In the monoclinic and orthorhombic asymmetric units one molecule contained iron and an NADP molecule, while the other did not. The tetragonal crystals lacked both iron and NADP. The structure is very similar to that of the FeADH from T. maritima (average r.m.s. difference for Cα atoms of 1.8 Å for 341 aligned atoms). The iron, which is internally sequestered, is bound entirely by amino acids from one domain: three histidines and one aspartic acid. The coenzyme is in an extended conformation, a feature that is common to the large superfamily of NADH-dependent dehydrogenases that share a classical nucleotide-binding domain. A long broad tunnel passes entirely through the enzyme between the two domains, completely encapsulating the coenzyme.

In the course of an earlier investigation into the crystallization of proteins based on the addition of intermolecular ligands, the Kunitz type trypsin inhibitor from soybean (SBTI) was crystallized as a complex with 1,5-disulfonylnaphthalene (ligand library 21D). The two molecules within the asymmetric unit of the monoclinic crystals are related by a near-exact NCS 2-fold axis and have essentially the same conformation as was found for them in previous analyses. The protein dimer is maintained through electrostatic interactions with the sulfonyl groups of four 1,5-disulfonylnaphthalene molecules, which are bound in pairs at the dimer interface and involve symmetrical pairs of arg 30 and arg 47 side chains of each SBTI molecule. The binding of the 1,5-disulfonylnaphthalene molecules at the interface is disordered in that only a coordinated single pair of 1,5-disulfonylnaphthalene molecules can be bound at any one time. There are also molecules of the MES buffer present in the crystal structure that are responsible for additional lattice interactions. This crystal structure provides a remarkable demonstration of how unusual, nonphysiologic, small molecules can create symmetry and promote specific lattice interactions within protein crystals.

Human endothelin is a 21-amino-acid polypeptide, constrained by two intra-chain disulfide bridges, that is made by endothelial cells. It is the most potent vasoconstrictor in the body and is crucially important in the regulation of blood pressure. It plays a major role in a host of medical conditions, including hypertension, diabetes, stroke and cancer. Endothelin was crystallized 28 years ago in the putative space group P6122, but the structure was never successfully solved by X-ray diffraction. Using X-ray diffraction data from 1992, the structure has now been solved. Assuming a unit cell belonging to space group P61 and a twin fraction of 0.28, a solution emerged with two, almost identical, closely associated molecules in the asymmetric unit. Although the data extended to beyond 1.8 Å resolution, a model containing 25 waters was refined to 1.85 Å resolution with an R of 0.216 and an Rfree of 0.284. The disulfide-constrained `core' of the molecule, amino-acid residues 1-15, has a main-chain conformation that is essentially the same as endothelin when bound to its receptor, but many side-chain rotamers are different. The carboxy-terminal `tail' comprising amino-acid residues 16-21 is extended as when receptor-bound, but it exhibits a different conformation with respect to the `core'. The dimer that comprises the asymmetric unit is maintained almost exclusively by hydrophobic interactions and may be stable in an aqueous medium.

Human apolipoprotein C1 (APOC1) is a 57 amino acid long polypeptide that, through its potent inhibition of cholesteryl ester transferase protein, helps regulate the transfer of lipids between lipid particles. We have now determined the structure of APOC1 in four crystal forms by X-ray diffraction. A molecule of APOC1 is a single, slightly bent, α-helix having 13–14 turns and a length of about 80 Å. APOC1 exists as a dimer, but the dimers are not the same in the four crystals. In two monoclinic crystals, two helices closely engage one another in an antiparallel fashion. The interactions between monomers are almost entirely hydrophobic with sparse electrostatic complements. In the third monoclinic crystal, the two monomers spread at one end of the dimer, like a scissor opening, and, by translation along the crystallographic a axis, form a continuous, contiguous sheet through the crystal. In the orthorhombic crystals, two molecules of APOC1 are related by a noncrystallographic 2-fold axis to create an arc of about 120 Å length. This symmetrical dimer utilizes interactions not present in dimers of the monoclinic crystals. Versatility of APOC1 monomer association shown by these crystals is suggestive of physiological function.

Bovine β-lactoglobulin was crystallized from 3 M NaCl buffered at pH 3.8 with sodium citrate as thick hexagonal prisms of greater than 1 mm in edge length. Analyses of the X-ray diffraction intensities using three different current algorithms were unanimous in specifying the space group to be P6322, with unit-cell dimensions a = b = 75.47, c = 140.79 Å. No progress could be made, however, towards an acceptable solution by molecular replacement using this symmetry. In the end, it was found that the true space group was C2221, a subgroup of P6322, with a = 65.89, b = 114.12, c = 140.51 Å, with the apparent 622 symmetry arising from an unusual threefold or tritohedral twinning. An assembly based on a model of the protein in another crystal form (PDB entry 1beb) containing three molecules in the asymmetric unit was refined to 2.3 Å resolution with a final R factor of 0.23 and Rfree of 0.26. NCS restraints were maintained throughout. For the most part, the molecules found in this crystal form are virtually the same as in PDB entry 1beb, although there are numerous local variations, particularly in loop elements, rotamer conformation differences and some alterations, including additions, at the termini.

Experiments were carried out on 15 different protein crystals with the objective of estimating the rates of penetration of dye molecules into the crystals. The dyes were in the molecular-weight range 250-1000 Da and the protein crystals were of dimensions of 0.7 mm or greater. Experiments were also conducted on protein crystals grown between glass cover slips (separation 200 µm) that restricted the direction of diffusion. The rate of penetration of dyes into protein crystals depends very much on the degree of association between the dye and protein molecules. Dye penetration was not consistent with pure diffusion when the affinity of the protein for the dye was significant, and this was frequent. Penetration rates were less dependent on factors such as the molecular weight of the dye or the diffusion direction. For weakly interacting protein crystal/dye combinations, penetration was a fair measure of diffusivity and the observed rates were in the range 60-100 µm h-1. For strongly interacting combinations, the rates of penetration were of the order of 15-30 µm h-1.

Protein crystals, otherwise usually colorless, can be stained a variety of hues by saturating them with dyes, by diffusion from the mother liquor or co-crystallization. The colors assumed by dyes are a function of chemical factors, particularly pH and redox potential. Protein crystals saturated with a pH sensitive dye, initially at one pH, can be exposed to the mother liquor at a second pH and the crystal will change color over time as H3O+ ions diffuse through the crystal. This allows diffusion rates of H3O+ through the crystal to be measured. Diffusion fronts are often clearly delineated. Similar experiments can be carried out with redox sensitive dyes by adding reductants, such as ascorbic acid or dithionite, or oxidants such as H2O2, to the crystal’s mother liquor. Presented here are a number of experiments using pH or redox sensitive dye-saturated protein crystals, and some experiments using double dye, sequential redox–pH changes.

The β subunit of bovine luteinizing hormone (LH) was crystallized and its structure solved to 3.15 ​Å resolution by molecular replacement using human chorionic gonadotropin (hCG) β subunit as search model. The asymmetric unit contains two copies of the β subunit that are related by a non-crystallographic symmetry (NCS) two-fold axis, both copies of which contain proteolytic cleavages after amino acid 100. It is noteworthy that the oligosaccharide moieties covalently attached at asparagine 13 were particularly pronounced in the electron density, allowing seven sugar residues to be defined. The α subunit of LH, which is common to all glycosylated gonadotropin hormones, was placed by superposition of hCG on the LH beta subunits, thereby yielding a model for the intact hormone.

It was found that the crystals of at least a dozen different proteins could be thoroughly stained to an intense color with a panel of dyes. Many, if not most, of the stained protein crystals retained the dyes almost indefinitely when placed in large volumes of dye-free mother liquor. Dialysis experiments showed that most of the dyes that were retained in crystals also bound to the protein when free in solution; less frequently, some dyes bound only in the crystal. The experiments indicated a strong association of the dyes with the proteins. Four protein crystals were investigated by X-ray diffraction to ascertain the mode of binding. These were crystals of lysozyme, thaumatin, trypsin inhibited with benzamidine and satellite tobacco mosaic virus. In 30 X-ray analyses of protein crystal–dye complexes, in only three difference Fourier maps was any difference electron density present that was consistent with the binding of dye molecules, and even in these three cases (thaumatin plus thioflavin T, xylene cyanol and m -cresol purple) the amount of dye observed was inadequate to explain the intense color of the crystals. It was concluded that the dye molecules, which are clearly inside the crystals, are disordered but are paradoxically tightly bound to the protein. It is speculated that the dyes, which exhibit large hydrophobic cores and peripheral charged groups, may interact with the crystalline proteins in the manner of conventional detergents.