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The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1–3, but also revealed how quickly viral escape can curtail effective options4, 5. With the emergence of the SARS-CoV-2 Omicron variant in late 2021, many clinically used antibody drug products lost potency, including EvusheldTMand its constituent, cilgavimab4, 6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies with a known clinical profile to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign COV2-2130 to rescue in vivo efficacy against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the contemporaneously dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and many variants of concern that subsequently emerged, and provides protectionin vivoagainst the strains tested, WA1/2020, BA.1.1, and BA.5. Deep mutational scanning of tens of thousands pseudovirus variants reveals 2130-1-0114-112 improves broad potency without incurring additional escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Because our approach is computationally driven, not requiring experimental iterations or pre-existing binding data, it could enable rapid response strategies to address escape variants or pre-emptively mitigate escape vulnerabilities.

Legionella is a genus of ubiquitous environmental pathogens found in freshwater systems, moist soil, and composted materials. More than four decades of Legionella research has provided important insights into Legionella pathogenesis. Although standard commercial microscopes have led to significant advances in understanding Legionella pathogenesis, great potential exists in the deployment of more advanced imaging techniques to provide additional insights. The lattice light sheet microscope (LLSM) is a recently developed microscope for 4D live cell imaging with high resolution and minimum photo-damage. We built a LLSM with an improved version for the optical layout with two path-stretching mirror sets and a novel reconfigurable galvanometer scanner (RGS) module to improve the reproducibility and reliability of the alignment and maintenance of the LLSM. We commissioned this LLSM to study Legionella pneumophila infection with a tailored workflow designed over instrumentation, experiments, and data processing methods. Our results indicate that Legionella pneumophila infection is correlated with a series of morphological signatures such as smoothness, migration pattern and polarity both statistically and dynamically. Our work demonstrates the benefits of using LLSM for studying long-term questions in bacterial infection. Our free-for-use modifications and workflow designs on the use of LLSM system contributes to the adoption and promotion of the state-of-the-art LLSM technology for both academic and commercial applications.

Legionella is a genus of ubiquitous environmental pathogens found in freshwater systems, moist soil, and composted materials. More than four decades of Legionella research has provided important insights into Legionella pathogenesis [1]. Although standard commercial microscopes have led to significant advances in understanding Legionella pathogenesis [2,3], great potential exists in the deployment of more advanced imaging techniques to provide additional insights. The Lattice Light Sheet Microscope (LLSM) is a recently developed microscope for 4D live cell imaging with high resolution and minimum photo-damage [4]. We built a LLSM with an improved version for the optical layout with two path-stretching mirror sets and a novel Reconfigurable Galvanometer Scanner (RGS) module to improve the reproducibility and reliability of the alignment and maintenance of the LLSM. We commissioned this LLSM to study Legionella pneumophila infection with a tailored workflow designed over instrumentation, experiments, and data processing methods. Our results indicate that Legionella pneumophila infection is correlated with a series of morphological signatures such as smoothness, migration pattern and polarity both statistically and dynamically. Our work demonstrates the benefits of using LLSM for studying long-term questions in bacterial infection. Our free-for-use modifications and workflow designs on the use of LLSM system contributes to the adoption and promotion of the state-of-the-art LLSM technology for both academic and commercial applications.

SummaryRapid assessment of whether a pandemic pathogen may have increased transmissibility or be capable of evading existing vaccines and therapeutics is critical to mounting an effective public health response. Over the period of seven days, we utilized rapid computational prediction methods to evaluate potential public health implications of the emerging SARS-CoV-2 Omicron variant. Specifically, we modeled the structure of the Omicron variant, examined its interface with human angiotensin converting enzyme 2 (ACE-2) and evaluated the change in binding affinity between Omicron, ACE-2 and publicly known neutralizing antibodies. We also compared the Omicron variant to known Variants of Concern (VoC). Seven of the 15 Omicron mutations occurring in the spike protein receptor binding domain (RBD) occur at the ACE-2 cell receptor interface, and therefore may play a critical role in enhancing binding to ACE-2. Our estimates of Omicron RBD-ACE-2 binding affinities indicate that at least two of RBD mutations, Q493R and N501Y, contribute to enhanced ACE-2 binding, nearly doubling delta-delta-G (ddG) free energies calculated for other VoC’s. Binding affinity estimates also were calculated for 55 known neutralizing SARS-CoV-2 antibodies. Analysis of the results showed that Omicron substantially degrades binding for more than half of these neutralizing SARS-CoV-2 antibodies, and for roughly 10 times as many of the antibodies than the currently dominant Delta variant. This early study lends support to use of rapid computational risk assessments to inform public health decision-making while awaiting detailed experimental characterization and confirmation.

Structure-based Deep Fusion models were recently shown to outperform several physics- and machine learning-based protein-ligand binding affinity prediction methods. As part of a multi-institutional COVID-19 pandemic response, over 500 million small molecules were computationally screened against four protein structures from the novel coronavirus (SARS-CoV-2), which causes COVID-19. Three enhancements to Deep Fusion were made in order to evaluate more than 5 billion docked poses on SARS-CoV-2 protein targets. First, the Deep Fusion concept was refined by formulating the architecture as one, coherently backpropagated model (Coherent Fusion) to improve binding-affinity prediction accuracy. Secondly, the model was trained using a distributed, genetic hyper-parameter optimization. Finally, a scalable, high-throughput screening capability was developed to maximize the number of ligands evaluated and expedite the path to experimental evaluation. In this work, we present both the methods developed for machine learning-based high-throughput screening and results from using our computational pipeline to find SARS-CoV-2 inhibitors.

With the nexus of super computing and the biotech revolution, it seems an era of predictive biology through systems biology may be at hand. Modern omics capabilities enable examination of the state of biological system in exquisite detail. The genome, transcriptome, proteome, and metabolome may all be largely knowable, at least for some model systems, providing a basis for modeling and simulation of molecular mechanisms, or pathways, that could capture a biological system's emergent properties. However, there are significant challenges remaining that impede the realization of this vision, perhaps the most significant being the missing functional annotation of genes and gene products. For even the most well-studied organisms as much as a third of called genes for a given genome are not annotated and more than half may be tenuous. Homology inferred from sequence similarity is the basis for much of genome annotation. Homology inferred from structural similarity could be a powerful complement to sequence-based annotation methods. Structural biology or structural informatics can be used to assign molecular function and may have increasing utility with the rapid growth of gene sequence databases and emerging methods for structure determination, like structure prediction based on coevolution. Here we describe tools and provide example cases using structural similarity at the level of quaternary structure, domain content, domain topology, and small 3D motifs to infer homology and posit function. Ultimately annotation by similarity, be it 3D structure homology or more classically primary sequence homology, must be founded by accurate annotation of one ortholog in the group-understanding every function encoded by a genome remains a major challenge to life science.

With the nexus of super computing and the biotech revolution, it seems an era of predictive biology through systems biology may be at hand. Modern omics capabilities enable examination of the state of biological system in exquisite detail. The genome, transcriptome, proteome, and metabolome may all be largely knowable, at least for some model systems, providing a basis for modeling and simulation of molecular mechanisms, or pathways, that could capture a biological system's emergent properties. However, there are significant challenges remaining that impede the realization of this vision, perhaps the most significant being the missing functional annotation of genes and gene products. For even the most well-studied organisms as much as a third of called genes for a given genome are not annotated and more than half may be tenuous. Homology inferred from sequence similarity is the basis for much of genome annotation. Homology inferred from structural similarity could be a powerful complement to sequence-based annotation methods. Structural biology or structural informatics can be used to assign molecular function and may have increasing utility with the rapid growth of gene sequence databases and emerging methods for structure determination, like structure prediction based on coevolution. Here we describe tools and provide example cases using structural similarity at the level of quaternary structure, domain content, domain topology, and small 3D motifs to infer homology and posit function. Ultimately annotation by similarity, be it 3D structure homology or more classically primary sequence homology, must be founded by accurate annotation of one ortholog in the group-understanding every function encoded by a genome remains a major challenge to life science.

IUCrJ 5(1), 103 - 117 (2018). doi:10.1107/S2052252517017043, Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals., Published by IUCr, Chester

This work demonstrates the use of polymer thin films and graphene to support and maintain the hydration of protein microcrystals on fixed targets for serial femtosecond crystallography at X-ray free-electron lasers. Rapid encystment protein (REP24) provides a benchmark for this encapsulation approach., For serial femtosecond crystallography at X-ray free-electron lasers, which entails collection of single-pulse diffraction patterns from a constantly refreshed supply of microcrystalline sample, delivery of the sample into the X-ray beam path while maintaining low background remains a technical challenge for some experiments, especially where this methodology is applied to relatively low-ordered samples or those difficult to purify and crystallize in large quantities. This work demonstrates a scheme to encapsulate biological samples using polymer thin films and graphene to maintain sample hydration in vacuum conditions. The encapsulated sample is delivered into the X-ray beam on fixed targets for rapid scanning using the Roadrunner fixed-target system towards a long-term goal of low-background measurements on weakly diffracting samples. As a proof of principle, we used microcrystals of the 24 kDa rapid encystment protein (REP24) to provide a benchmark for polymer/graphene sandwich performance. The REP24 microcrystal unit cell obtained from our sandwiched in-vacuum sample was consistent with previously established unit-cell parameters and with those measured by us without encapsulation in humidified helium, indicating that the platform is robust against evaporative losses. While significant scattering from water was observed because of the sample-deposition method, the polymer/graphene sandwich itself was shown to contribute minimally to background scattering.

Crystals 10(10), 886 (2020). doi:10.3390/cryst10100886, Nanolipoprotein particles (NLPs), also called ���nanodiscs���, are discoidal particles with a patch of lipid bilayer corralled by apolipoproteins. NLPs have long been of interest due to both their utility as membrane-model systems into which membrane proteins can be inserted and solubilized and their physiological role in lipid and cholesterol transport via high-density lipoprotein (HDL) and low-density lipoprotein (LDL) maturation, which are important for human health. Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) is a powerful approach for structural biology of membrane proteins, which are traditionally difficult to crystallize as large single crystals capable of producing high-quality diffraction suitable for structure determination. To facilitate understanding of the specific role of two apolipoprotein/lipid complexes, ApoA1 and ApoE4, in lipid binding and HDL/LDL particle maturation dynamics, and to develop new SFX methods involving NLP membrane protein encapsulation, we have prepared and crystallized homogeneous populations of ApoA1 and ApoE4 NLPs. Crystallization of empty NLPs yields semi-ordered objects that appear crystalline and give highly anisotropic and diffuse X-ray diffraction, similar to fiber diffraction. Several unit cell parameters were approximately determined for both NLPs from these measurements. Thus, low-background, sample conservative methods of delivery are critical. Here we implemented a fixed target sample delivery scheme utilizing the Roadrunner fast-scanning system and ultra-thin polymer/graphene support films, providing a low-volume, low-background approach to membrane protein SFX. This study represents initial steps in obtaining structural information for ApoA1 and ApoE4 NLPs and developing this system as a supporting scaffold for future structural studies of membrane proteins crystalized in a native lipid environment., Published by MDPI, Basel

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introduction via a translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Z plastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low-Z fixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

Bragg diffraction achieved from two-dimensional protein crystals using femtosecond X-ray laser snapshots is presented., X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

Here we present a perspective on a range of practical uses of structural genomics for mutagen research. Structural genomics is an overloaded term and requires some definition to bound the discussion; we give a brief description of public and private structural genomics endeavors, along with some of their objectives, their activities, their capabilities, and their limitations. We discuss how structural genomics might impact mutagen research in three different scenarios: at a structural genomics center, at a lab with modest resources that also conducts structural biology research, and at a lab that is conducting mutagen research without in-house experimental structural biology. Applications span functional annotation of single genes or SNP, to constructing gene networks and pathways, to an integrated systems biology approach. Structural genomics centers can take advantage of systems biology models to target high value targets for structure determination and in turn extend systems models to better understand systems biology diseases or phenomenon. Individual investigator run structural biology laboratories can collaborate with structural genomics centers, but can also take advantage of technical advances and tools developed by structural genomics centers and can employ a structural genomics approach to advancing biological understanding. Individual investigator-run non-structural biology laboratories can also collaborate with structural genomics centers, possibly influencing targeting decisions, but can also use structure based annotation tools enabled by the growing coverage of protein fold space provided by structural genomics. Better functional annotation can inform pathway and systems biology models.

The crystal structure of the urease gamma subunit (UreA) from Mycobacterium tuberculosis, Rv1848, has been determined at 1.8 A resolution. The asymmetric unit contains three copies of Rv1848 arranged into a homotrimer that is similar to the UreA trimer in the structure of urease from Klebsiella aerogenes. Small-angle X-ray scattering experiments indicate that the Rv1848 protein also forms trimers in solution. The observed homotrimer and the organization of urease genes within the M. tuberculosis genome suggest that M. tuberculosis urease has the (alphabetagamma)(3) composition observed for other bacterial ureases. The gamma subunit may be of primary importance for the formation of the urease quaternary structure.

The LcrV protein is a multifunctional virulence factor and protective antigen of the plague bacterium and is generally conserved between the epidemic strains of Yersinia pestis. We investigated the diversity in the LcrV sequences among non-epidemic Y. pestis strains which have a limited virulence in selected animal models and for humans. Sequencing of lcrV genes from 19 Y. pestis strains belonging to different phylogenetic groups (“subspecies”) showed that the LcrV proteins possess four major variable hotspots at positions 18, 72, 273, and 324–326. These major variations, together with other minor substitutions in amino acid sequences, allowed us to classify the LcrV alleles into five sequence types (A-E). We observed that the strains of different Y. pestis “subspecies” can have the same type of LcrV, including that conserved in epidemic strains, and different types of LcrV can exist within the same natural plague focus. Therefore, the phenomenon of “selective virulence” characteristic of the strains of the microtus biovar is unlikely to be the result of polymorphism of the V antigen. The LcrV polymorphisms were structurally analyzed by comparing the modeled structures of LcrV from all available strains. All changes except one occurred either in flexible regions or on the surface of the protein, but local chemical properties (i.e. those of a hydrophobic, hydrophilic, amphipathic, or charged nature) were conserved across all of the strains. Polymorphisms in flexible and surface regions are likely subject to less selective pressure, and have a limited impact on the structure. In contrast, the substitution of tryptophan at position 113 with either glutamic acid or glycine likely has a serious influence on the regional structure of the protein, and these mutations might have an effect on the function of LcrV. The polymorphisms at positions 18, 72 and 273 were accountable for differences in the oligomerization of LcrV.

To better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly.

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Delta1-49 apolipoprotein A-I fragment (Delta49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR --M transition. Importantly the functional bR was solubilized in discoidal bR.NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Delta49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.

Spontaneous interaction of purified apolipoproteins and phospholipids results in formation of lipoprotein particles with nanometer-sized dimensions; we refer to these assemblies as nanolipoprotein particles or NLPs. These bilayer constructs can serve as suitable mimetics of biological membranes and are fully soluble in aqueous environments. We made NLPs from dimyristoylphospatidylcholine (DMPC) in combination with each of four different apolipoproteins: apoA-I, Delta-apoA-I fragment, apoE4 fragment, and apolipophorin III (apoLp-III) from the silk moth B. mori. Predominately discoidal in shape, these particles have diameters between 10 and 20 nm, share uniform heights between 4.5 and 5 nm, and can be produced in yields ranging between 40 and 60%. The particular lipoprotein, the lipid to lipoprotein ratio, and the assembly parameters determine the size and homogeneity of nanolipoprotein particles and indicate that apoA-I NLP preparations are smaller than the larger apoE422K and apoLp-III NLP preparations.

An automated platform for low-volume, highthroughput protein crystallization by the sitting drop vapor diffusion has been developed. This platform consists of a novel Society of Biomolecular Screening standard microplate technology, standard automation liquid handling equipment, a software to design customizable, random crystallization buffers, an in-house designed scanner, a crystal recognition software, and Laboratory Information Management Systems. These technologies are designed to eliminate obstacles to the production of protein crystals and to address a number of critical issues faced by crystallography laboratories, namely precision and accuracy, ease of integration and use, speed, and cost.

The three-dimensional structure of Rv2607, a putative pyridoxine 5'-phosphate oxidase (PNPOx) from Mycobacterium tuberculosis, has been determined by X-ray crystallography to 2.5 A resolution. Rv2607 has a core domain similar to known PNPOx structures with a flavin mononucleotide (FMN) cofactor. Electron density for two FMN at the dimer interface is weak despite the bright yellow color of the protein solution and crystal. The shape and size of the putative binding pocket is markedly different from that of members of the PNPOx family, which may indicate some significant changes in the FMN binding mode of this protein relative to members of the family.

Fluidigm Corp. released the Topaz™ 1.96 and 4.96 crystallization chips in the fall of 2004. Topaz 1.96 and 4.96 are the latest evolution of Fluidigm’s microfluidics crystallization technologies that enable ultra-low-volume rapid screening for macromolecular crystallization. Topaz 1.96 and 4.96 are similar to each other but represent a major redesign of the Topaz system and have substantially improved ease of automation and ease of use, improved efficiency and even further reduced the amount of material needed. With the release of the new Topaz system, Fluidigm continues to set the standard in low-volume crystallization screening, which is having an increasing impact in the field of structural genomics and more generally in structural biology. It is likely that further optimization and increased utility of the Topaz crystallization system will emerge. It is also probable that further innovation and the emergence of competing technologies will be seen.

Clostridium botulinum neurotoxins (BoNTs), the most potent toxins known, disrupt neurotransmission through proteolysis of proteins involved in neuroexocytosis. The light chains of BoNTs are unique zinc proteases that have stringent substrate specificity and require exceptionally long substrates. We have determined the crystal structure of the protease domain from BoNT serotype A (BoNT/A). The structure reveals a homodimer in a product-bound state, with loop F242–V257 from each monomer deeply buried in its partner's catalytic site. The loop, which acts as a substrate, is oriented in reverse of the canonical direction for other zinc proteases. The Y249–Y250 peptide bond of the substrate loop is hydrolyzed, leaving the Y249 product carboxylate coordinated to the catalytic zinc. From the crystal structure of the BoNT/A protease, detailed models of noncanonical binding and proteolysis can be derived which we propose are also consistent with BoNT/A binding and proteolysis of natural substrate synaptosome-associated protein of 25 kDa (SNAP-25). The proposed BoNT/A substrate-binding mode and catalytic mechanism are markedly different from those previously proposed for the BoNT serotype B.

The Mycobacterium tuberculosis rmlC gene encodes dTDP-4-­keto-6-deoxyglucose epimerase, the third enzyme in the M. tuberculosis dTDP-l-rhamnose pathway which is essential for mycobacterial cell-wall synthesis. Because it is structurally unique, highly substrate-specific and does not require a cofactor, RmlC is considered to be the most promising drug target in the pathway, and the M. tuberculosis rmlC gene was selected in the initial round of TB Structural Genomics Consortium targets for structure determination. The 1.7 A native structure determined by the consortium facilities is reported and implications for in silico screening of ligands for structure-guided drug design are discussed.

Anticipating a continuing increase in the number of structures solved by molecular replacement in high-throughput crystallography and drug-discovery programs, a user-friendly web service for automated molecular replacement, map improvement, bias removal and real-space correlation structure validation has been implemented. The service is based on an efficient bias-removal protocol, Shake&wARP, and implemented using EPMR and the CCP4 suite of programs, combined with various shell scripts and Fortran90 routines. The service returns improved maps, converted data files and real-space correlation and B-factor plots. User data are uploaded through a web interface and the CPU-intensive iteration cycles are executed on a low-cost Linux multi-CPU cluster using the Condor job-queuing package. Examples of map improvement at various resolutions are provided and include model completion and reconstruction of absent parts, sequence correction, and ligand validation in drug-target structures.

A structure of native concanavalin A (ConA), a hardy perennial of structural biology, has been determined in a dimeric crystal form at a resolution of 1.56 A (space group C222(1); unit-cell parameters a = 118.70, b = 101.38, c = 111.97 A; two molecules in the asymmetric unit). The structure has been refined to an R(free) of 0.206 (R = 0.178) after iterative model building and phase-bias removal using ShakewARP. Correspondence between calculated water-tyrosine interactions and experimentally observed structures near the saccharide-binding site suggests that the observed interactions between Tyr12 and water in various crystal forms are to be expected and are not unique to the presence of an active site. The present structure differs from previously reported atomic resolution structures of ConA in several regions and extends insight into the conformational flexibility of this molecule. Furthermore, this third, low-temperature, structure of ConA in a different crystal form, independently refined using powerful model-bias removal techniques, affords the opportunity to revisit assessment of accuracy and precision in high- or atomic resolution protein structures. It is illustrated that several precise structures of the same molecule can differ substantially in local detail and users of crystallographic models are reminded to consider the potential impact when interpreting structures. Suggestions on how to effectively represent ensembles of crystallographic models of a given molecule are provided.

Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essential to the pathogen's long term intracellular survival in the amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1′ recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells.

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

In an effort to objectively compare the efficiency of protein crystallization screening techniques, a probability model of sampling efficiency is developed and used to calculate sampling efficiencies from experimental data. Three typical sampling protocols (grid screening, footprint screening, and random screening) are used to crystallize each of five proteins (Phospholipase A 2 , Thaumatin, Catalase, Lysozyme, and Ribonuclease B). For each of the three sampling protocols, experiments are chosen from a large set of possible experiments generated by systematic combination of a number of parameters common in crystallization screens. Software has been developed to generate and select from the combinations with each of the three sampling protocols examined in this study. The protocols differ only in the order samples are chosen from the set of possible combinations. Random sampling is motivated by the “Incomplete Factorial” screen (Carter and Carter, J. Biol. Chem. 254 (1979) 12 219); sampling with subsets of four is motivated by the “Footprint” screen (Stura et al., J. Crystal Growth 122 (1992) 273) and sampling with subsets of twenty-four is motivated by the “Grid” screen (McPherson, Prepartion and Analysis of Protein Crystals, Wiley, New York, 1982). For the five proteins examined, random sampling has the greatest average efficiency. Additional benefits of random sampling are discussed.

The major human abasic endonuclease, Ape1, is an essential DNA repair enzyme that initiates the removal of apurinic/apyrimidinic sites from DNA, excises 3′ replication-blocking moieties, and modulates the DNA binding activity of several transcriptional regulators. We have determined the X-ray structure of the full-length human Ape1 enzyme in two new crystal forms, one at neutral and one at acidic pH. The new structures are generally similar to the previously determined structure of a truncated Ape1 protein, but differ in the conformation of several loop regions and in spans of residues with weak electron density. While only one active-site metal ion is present in the structure determined at low pH, the structure determined from a crystal grown at the pH optimum of Ape1 nuclease activity, pH 7.5, has two metal ions bound 5 A apart in the active site. Enzyme kinetic data indicate that at least two metal-binding sites are functionally important, since Ca 2+ exhibits complex stimulatory and inhibitory effects on the Mg 2+ -dependent catalysis of Ape1, even though Ca 2+ itself does not serve as a cofactor. In conjunction, the structural and kinetic data suggest that Ape1 catalyzes hydrolysis of the DNA backbone through a two metal ion-mediated mechanism.

An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.

The paradigm that T cells recognize peptide antigens presented by major histocompatibility complex class I and class II molecules has been a guiding principle in the development of immunology. Here, Steven Porcelli and colleagues review studies that extend this paradigm by showing that CD1 proteins are a separate lineage of antigen-presenting molecules with unusually hydrophobic ligand-binding grooves that present nonpeptide lipid and glycolipid antigens to T cells.

Three crystal forms of Naja naja naja phospholipase A 2 were discovered through random crystallization screening, including two heretofore uncharacterized forms. The crystallization conditions for both of these novel crystal forms are Ca 2+ -free whereas previously reported conditions include Ca 2+ . One of the new crystal forms has a cubic lattice in the space group P 2 1 3 ( a = b = c =69.24 A), the other has an orthorhombic lattice in the space group P 2 1 2 1 2 1 ( a =67.22 A, b =73.48 A, c =87.52 A) and a previously characterized crystal belong to the tetragonal space group P 4 3 2 1 2 ( a = b =88.6 A, c =107.4 A). The structure from the cubic crystal form has been determined to 1.8 A and refined to an R -factor of 17% while the structure from the orthorhombic form has been determined to 2.65 A and has been refined to an R -factor of 21%. The determination of the cubic structure extends the resolution to which structures of this molecule have been determined from 2.3 A to 1.8 A. The two newly determined structures, in combination with the previously determined structure, generate an informative structural ensemble from which structural changes due to Ca 2+ , which is required for catalysis, and the effect of crystal contacts on side-chain conformations and oligomeric association can be inferred. Both of the newly determined structures reveal a trimeric oligomer as observed in the tetragonal structure; this appears to be a unique feature of the Naja naja naja enzyme.

Transferred NOE experiments have been carried out on cobra venom (Naja naja naja) phospholipase A2 (PLA2) with substrate analogues which serve as potent inhibitors. 1-(Hexylthio)-2-(hexanoylamino)-1,2-dideoxy-sn-glycero-3-pho sphoethanolamine (PE) and the corresponding phosphocholine analogue (PC) are water-soluble, short-chain, nonhydrolyzable substrate analogues which bind tightly to the enzyme. Because they are small compounds and monomeric in solution, NOEs develop inefficiently in the absence of enzyme. Thus, the PLA2/inhibitor system is ideal for analyzing transferred NOEs. The experiments are carried out under conditions that are optimal for catalysis, pH 7.5 in the presence of 2 mM CaCl2. The data show the inhibitor conformation in the catalytic site of cobra PLA2 in solution. The effect of the thioether in the sn-1 chain on the chemical shift dispersion of the methylene protons allowed for chain-specific assignments and detailed conformational analysis. Both inhibitors adopt a PLA2-bound conformation in which the end of the sn-2 chain is within 5 A of the alpha-methylene of the sn-1 chain. In addition, intermolecular contact points between the inhibitor and the enzyme were identified by NOEs.

The proposed research was motivated by an interest in understanding Y. pestis virulence mechanisms and bacteria cell-cell communication. It is expected that a greater understanding of virulence mechanisms will ultimately lead to biothreat countermeasures and novel therapeutics. Y. pestis is the etiological agent of plague, the most devastating disease in human history. Y. pestis infection has a high mortality rate and a short incubation before mortality. There is no widely available and effective vaccine for Y. pestis and multi-drug resistant strains are emerging. Y. pestis is a recognized biothreat agent based on the wide distribution of the bacteria in research laboratories around the world and on the knowledge that methods exist to produce and aerosolize large amounts of bacteria. We hypothesized that cell-cell communication via signaling molecules, or quorum sensing, by Y. pestis is important for the regulation of virulence factor gene expression during host invasion, though a causative link had never been established. Quorum sensing is a mode of intercellular communication which enables orchestration of gene expression for many bacteria as a function of population density and available evidence suggests there may be a link between quorum sensing and regulation of Y. pesits virulence. Several pathogenic bacteria have been shown to regulate expression of virulence factor genes, including genes encoding type III secretion, via quorum sensing. The Y. pestis genome encodes several cell-cell signaling pathways and the interaction of at least three of these are thought to be involved in one or more modes of host invasion. Furthermore, Y. pestis gene expression array studies carried out at LLNL have established a correlation between expression of known virulence factors and genes involved in processing of the AI-2 quorum sensing signal. This was a basic research project that was intended to provide new insights into bacterial intercellular communication and how it is used to regulate virulence in Y. pestis. It is known that many bacteria use intercellular signaling molecules to orchestrate gene expression and cellular function. A fair amount is known about production and uptake of signaling molecules, but very little is known about how intercellular signaling regulates other pathways. Although several studies demonstrate that intercellular signaling plays a role in regulating virulence in other pathogens, the link between signaling and regulation of virulence has not been established. Very little work had been done directly with Y. pestis intercellular signaling apart from the work carried out at LLNL. The research we proposed was intended to both establish a causative link between AI-2 intercellular signaling and regulation of virulence in Y. pestis and elucidate the fate of the AI-2 signaling molecule after it is taken up and processed by Y. pestis. Elucidating the fate of AI-2 was expected to lead directly to the understanding of how AI-2 signal processing regulates other pathways as well as provide new insights in this direction.

BACKGROUND: Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs. METHODOLOGY: Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to >25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4 degrees C was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes. CONCLUSIONS: ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utility for biotechnological applications.

Heterogeneity is a fact that plagues the characterization and application of many self-assembled biological constructs. The importance of obtaining particle homogeneity in biological assemblies is a critical goal, as bulk analysis tools often require identical species for reliable interpretation of the results-indeed, important tools of analysis such as x-ray diffraction typically require over 90% purity for effectiveness. This issue bears particular importance in the case of lipoproteins. Lipid-binding proteins known as apolipoproteins can self assemble with liposomes to form reconstituted high density lipoproteins (rHDLs) or nanolipoprotein particles (NLPs) when used for biotechnology applications such as the solubilization of membrane proteins. Typically, the apolipoprotein and phospholipids reactants are self assembled and even with careful assembly protocols the product often contains heterogeneous particles. In fact, size polydispersity in rHDLs and NLPs published in the literature are frequently observed, which may confound the accurate use of analytical methods. In this article, we demonstrate a procedure for producing a pure, monodisperse NLP subpopulation from a polydisperse self-assembly using size exclusion chromatography (SEC) coupled with high resolution particle imaging by atomic force microscopy (AFM). In addition, NLPs have been shown to self assemble both in the presence and absence of detergents such as cholate, yet the effects of cholate on NLP polydispersity and separation has not been systematically examined. Therefore, we examined the separation properties of NLPs assembled in both the absence and presence of cholate using SEC and native gel electrophoresis. From this analysis, NLPs prepared with and without cholate showed particles with well defined diameters spanning a similar size range. However, cholate was shown to have a dramatic affect on NLP separation by SEC and native gel electrophoresis. Furthermore, under conditions where different sized NLPs were not sufficiently separated or purified by SEC, AFM was used to deconvolute the elution pattern of different sized NLPs. From this analysis we were able to purify an NLP subpopulation to 90% size homogeneity by taking extremely fine elutions from the SEC. With this purity, we generate high quality NLP crystals that were over 100 microm in size with little precipitate, which could not be obtained utilizing the traditional size exclusion techniques. This purification procedure and the methods for validation are broadly applicable to other lipoprotein particles.

Nanolipoprotein particles (NLPs) are nanometer-sized, discoidal particles that self-assemble from purified apolipoprotein and phospholipid. Their size and facile functionalization suggest potential application of NLPs as platforms for the presentation and delivery of recombinant proteins. To this end, we investigated incorporation of nickel-chelating lipids into NLPs (NiNLPs) and subsequent sequestration of polyhistidine (His)-tagged proteins. From initial lipid screens for NLP formation, the two phospholipids DMPC and DOPC were identified as suitable bulk lipids for incorporation of the nickel-chelating lipid DOGS-NTA-Ni into NLPs, and NiNLPs were successfully formed with varying amounts of DOGS-NTA-Ni. NiNLPs consisting of 10% DOGS-NTA-Ni with 90% bulk lipid (either DMPC or DOPC) were thoroughly characterized by size exclusion chromatography (SEC), non-denaturing gradient gel electrophoresis (NDGGE), and atomic force microscopy (AFM). Three different His-tagged proteins were sequestered on NiNLPs in a nickel-dependent manner, and the amount of immobilized protein was contingent on the size and composition of the NiNLP.

Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell-cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring of amphipathic protein of defined diameter. The NLPs act as a platform for inserting, solubilizing and characterizing functional membrane proteins. NLP component proteins (apolipoproteins), as well as membrane proteins can be produced by either traditional cell-based or as discussed here, cell-free expression methodologies.

Microfluidic technologies enable a relatively new approach to macromolecular crystallization, but offer several significant advantages over more traditional techniques. Microfluidic devices provide significant savings in the amount of material required to complete a set of experiments, although recent innovations with vapor diffusion and microbatch methods have also greatly reduced their material requirements. When compared with these other methods, microfluidic approaches still consume 5-100x less material. In addition, comparisons in one set of experiments suggest that microfluidic free-interface diffusion may also offer substantially higher success rates than sitting drop vapor diffusion. Microfluidic methods also provide opportunities for experimental strategies involving testing multiple samples in parallel. When combined with randomized design of screening reagents, microfluidic devices provide a highly efficient method for sampling crystallization space. Commercial microfluidic crystallization chips have been in circulation for a number of years now and stable protocols for their use, tips and tricks, and data on their success and failure are now available.

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at approximately 14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.

Microfluidic technologies enable a relatively new approach to macromolecular crystallization, but offer several significant advantages over more traditional techniques. Microfluidic devices provide significant savings in the amount of material required to complete a set of experiments, although recent innovations with vapor diffusion and microbatch methods have also greatly reduced their material requirements. When compared with these other methods, microfluidic approaches still consume 5-100x less material. In addition, comparisons in one set of experiments suggest that microfluidic free-interface diffusion may also offer substantially higher success rates than sitting drop vapor diffusion. Microfluidic methods also provide opportunities for experimental strategies involving testing multiple samples in parallel. When combined with randomized design of screening reagents, microfluidic devices provide a highly efficient method for sampling crystallization space. Commercial microfluidic crystallization chips have been in circulation for a number of years now and stable protocols for their use, tips and tricks, and data on their success and failure are now available.

Tuberculosis (TB) infects one-third of the world population. Despite 50 years of available drug treatments, TB continues to increase at a significant rate. The failure to control TB stems in part from the expense of delivering treatment to infected individuals and from complex treatment regimens. Incomplete treatment has fueled the emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (Mtb). Reducing non-compliance by reducing the duration of chemotherapy will have a great impact on TB control. The development of new drugs that either kill persisting organisms, inhibit bacilli from entering the persistent phase, or convert the persistent bacilli into actively growing cells susceptible to our current drugs will have a positive effect. We are taking a multidisciplinary approach that will identify and characterize new drug targets that are essential for persistent Mtb. Targets are exposed to a battery of analyses including microarray experiments, bioinformatics, and genetic techniques to prioritize potential drug targets from Mtb for structural analysis. Our core structural genomics pipeline works with the individual laboratories to produce diffraction quality crystals of targeted proteins, and structural analysis will be completed by the individual laboratories. We also have capabilities for functional analysis and the virtual ligand screening to identify novel inhibitors for target validation. Our overarching goals are to increase the knowledge of Mtb pathogenesis using the TB research community to drive structural genomics, particularly related to persistence, develop a central repository for TB research reagents, and discover chemical inhibitors of drug targets for future development of lead compounds.