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\"For two centuries, travellers were amazed at the massive buildings found along the rivers that flow from the mountainous interior of Borneo. They concentrated hundreds of people under one roof, in the middle of empty rainforests. There was no practical necessity for this arrangement, and it remains a mystery. Peter Metcalf provides an answer by showing the historical context, using both oral histories and colonial records. The key factor was a pre-modern trading system that funneled rare and exotic jungle products to China via the ancient coastal city of Brunei. Meanwhile the elite manufactured goods traded upriver shaped the political and religious institutions of longhouse society. However, the apparent permanence of longhouses was an illusion. In historical terms, longhouse communities were both mobile and labile, and the patterns of ethnicity they created more closely resemble the contemporary world than any stereotype of 'tribal' societies\"--Provided by publisher.

Tough cubes Insect viruses that produce infectious polyhedra — crystals encapsulating thousands of virus particles — are widespread and important. The polyhedra microcrystals are remarkably stable, which can cause disease persistence, threatening silkworm cocoon harvests for instance. The molecular structure of one of these crystals has now been determined. This is a major technical feat in protein X-ray microcrystallography, as these viruses are the smallest protein crystals ever used to determine atomic structure. The study reveals robust polyhedra that could be useful as delivery capsules for biopesticides and for nanobiotechnology applications such as microarrays. Cypoviruses and baculoviruses are notoriously difficult to eradicate because the virus particles are embedded in micrometre-sized protein crystals called polyhedra 1 , 2 . The remarkable stability of polyhedra means that, like bacterial spores, these insect viruses remain infectious for years in soil. The environmental persistence of polyhedra is the cause of significant losses in silkworm cocoon harvests but has also been exploited against pests in biological alternatives to chemical insecticides 3 , 4 . Although polyhedra have been extensively characterized since the early 1900s 5 , their atomic organization remains elusive 6 . Here we describe the 2 Å crystal structure of both recombinant and infectious silkworm cypovirus polyhedra determined using crystals 5–12 micrometres in diameter purified from insect cells. These are the smallest crystals yet used for de novo X-ray protein structure determination 7 . We found that polyhedra are made of trimers of the viral polyhedrin protein and contain nucleotides. Although the shape of these building blocks is reminiscent of some capsid trimers, polyhedrin has a new fold and has evolved to assemble in vivo into three-dimensional cubic crystals rather than icosahedral shells. The polyhedrin trimers are extensively cross-linked in polyhedra by non-covalent interactions and pack with an exquisite molecular complementarity similar to that of antigen–antibody complexes. The resulting ultrastable and sealed crystals shield the virus particles from environmental damage. The structure suggests that polyhedra can serve as the basis for the development of robust and versatile nanoparticles for biotechnological applications 8 such as microarrays 9 and biopesticides 4 .

Infectious protein crystals are an essential part of the viral lifecycle for double-stranded DNA Baculoviridae and double-stranded RNA cypoviruses. These viral protein crystals, termed occlusion bodies or polyhedra, are dense protein assemblies that form a crystalline array, encasing newly formed virions. Here, using X-ray crystallography we determine the structure of a polyhedrin from Nudiviridae . This double-stranded DNA virus family is a sister-group to the baculoviruses, whose members were thought to lack occlusion bodies. The 70-year-old sample contains a well-ordered lattice formed by a predominantly α-helical building block that assembles into a dense, highly interconnected protein crystal. The lattice is maintained by extensive hydrophobic and electrostatic interactions, disulfide bonds, and domain switching. The resulting lattice is resistant to most environmental stresses. Comparison of this structure to baculovirus or cypovirus polyhedra shows a distinct protein structure, crystal space group, and unit cell dimensions, however, all polyhedra utilise common principles of occlusion body assembly. Viral occlusion bodies are robust protein crystals that encapsulate virions of some insect viruses. Here, the authors determine the nudivirus occlusion body structure and describe common principles of occlusion body structure.

The Japanese firefly squid Hotaru-ika ( Watasenia scintillans ) produces intense blue light from photophores at the tips of two arms. These photophores are densely packed with protein microcrystals that catalyse the bioluminescent reaction using ATP and the substrate coelenterazine disulfate. The squid is the only organism known to produce light using protein crystals. We extracted microcrystals from arm tip photophores and identified the constituent proteins using mass spectrometry and transcriptome libraries prepared from arm tip tissue. The crystals contain three proteins, wsluc1–3, all members of the ANL superfamily of adenylating enzymes. They share 19 to 21% sequence identity with firefly luciferases, which produce light using ATP and the unrelated firefly luciferin substrate. We propose that wsluc1–3 form a complex that crystallises inside the squid photophores and that in the crystal one or more of the proteins catalyses the production of light using coelenterazine disulfate and ATP. These results suggest that ANL superfamily enzymes have independently evolved in distant species to produce light using unrelated substrates.

The life cycle of Baculoviridae family insect viruses depends on the viral protein kinase, PK-1, to phosphorylate the regulatory protein, p6.9, to induce baculoviral genome release. Here, we report the crystal structure of Cydia pomenella granulovirus PK-1, which, owing to its likely ancestral origin among host cell AGC kinases, exhibits a eukaryotic protein kinase fold. PK-1 occurs as a rigid dimer, where an antiparallel arrangement of the αC helices at the dimer core stabilizes PK-1 in a closed, active conformation. Dimerization is facilitated by C-lobe:C-lobe and N-lobe:N-lobe interactions between protomers, including the domain-swapping of an N-terminal helix that crowns a contiguous β-sheet formed by the two N-lobes. PK-1 retains a dimeric conformation in solution, which is crucial for catalytic activity. Our studies raise the prospect that parallel, side-to-side dimeric arrangements that lock kinase domains in a catalytically-active conformation could function more broadly as a regulatory mechanism among eukaryotic protein kinases. The viral Protein Kinase-1 (PK-1) phosphorylates the regulatory protein p6.9, which facilitates baculoviral genome release. Here, the authors combine X-ray crystallography with biophysical and biochemical analyses as well as molecular dynamics simulations to characterize Cydia pomenella granulovirus PK-1, which forms a dimer with a parallel side-to-side arrangement of the kinase domains and furthermore, they provide insights into its catalytic mechanism and evolutionary relationships with other kinases.

Baculoviruses are ubiquitous insect viruses well known for their use as bioinsecticides, gene therapy vectors, and protein expression systems. Overexpression of recombinant proteins in insect cell culture utilizes the strong promoter of the polyhedrin gene. In infected larvae, the polyhedrin protein forms robust intracellular crystals called polyhedra, which protect encased virions for prolonged periods in the environment. Polyhedra are produced by two unrelated families of insect viruses, baculoviruses and cypoviruses. The atomic structure of cypovirus polyhedra revealed an intricate packing of trimers, which are interconnected by a projecting N-terminal helical arm of the polyhedrin molecule. Baculovirus and cypovirus polyhedra share nearly identical lattices, and the N-terminal region of the otherwise unrelated baculovirus polyhedrin protein sequence is also predicted to be α-helical. These results suggest homology between the proteins and a common structural basis for viral polyhedra. Here, we present the 2.2-Å structure of baculovirus polyhedra determined by x-ray crystallography from microcrystals produced in vivo. We show that the underlying molecular organization is, in fact, very different. Although both polyhedra have nearly identical unit cell dimensions and share I23 symmetry, the polyhedrin molecules are structurally unrelated and pack differently in the crystals. In particular, disulfide bonds and domain-swapped N-terminal domains stabilize the building blocks of baculovirus polyhedra and interlocking C-terminal arms join unit cells together. We show that the N-terminal projecting helical arms have different structural roles in baculovirus and cypovirus polyhedra and conclude that there is no structural evidence for a common evolutionary origin for both classes of polyhedra.

DsbC is one of five Escherichia coli proteins required for disulfide bond formation and is thought to function as a disulfide bond isomerase during oxidative protein folding in the periplasm. DsbC is a 2 × 23 kDa homodimer and has both protein disulfide isomerase and chaperone activity. We report the 1.9 Å resolution crystal structure of oxidized DsbC where both Cys-X-X-Cys active sites form disulfide bonds. The molecule consists of separate thioredoxin-like domains joined via hinged linker helices to an N-terminal dimerization domain. The hinges allow relative movement of the active sites, and a broad uncharged cleft between them may be involved in peptide binding and DsbC foldase activities.

The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitin-rich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides. Significance X-ray crystallography is a powerful approach for understanding the structure and function of biological macromolecules but is largely limited to molecules that form high-quality crystals in the laboratory. Here we present the structure of protein crystals that form naturally in virally infected insects and boost the insecticidal activity of oral pathogens. By proposing a mode of action for these virulence factors based on enzymes degrading chitin by oxidation, our findings may guide their use as synergetic additives to common bioinsecticides. They also reveal that these proteins assemble into ultra-stable crystals stabilized by a 3D network of covalent bonds, a unique strategy for achieving efficient protein crystallization in the complex environment of the cell.

To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses fromX-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm³ in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm³ in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.

The correct formation of disulfide bonds in the periplasm of Escherichia coli involves Dsb proteins, including two related periplasmic disulfide-bond isomerases, DsbC and DsbG. DsbD is a membrane protein required to maintain the functional oxidation state of DsbC and DsbG. In this work, purified proteins were used to investigate the interaction between DsbD and DsbC. A 131-residue N-terminal fragment of DsbD (DsbDα) was expressed and purified and shown to form a functional folded domain. Gel filtration results indicate that DsbDα is monomeric. DsbDα was shown to interact directly with and to reduce the DsbC dimer, thus increasing the isomerase activity of DsbC. The DsbC-DsbDα complex was characterized, and formation of the complex was shown to require the N-terminal dimerization domain of DsbC. These results demonstrate that DsbD interacts directly with full-length DsbC and imply that no other periplasmic components are required to maintain DsbC in the functional reduced state.