Structural features of sensory two component systems: a synthetic biology perspective.

All living organisms include a set of signaling devices that confer the ability to dynamically perceive and adapt to the fluctuating environment. Two-component systems are part of this sensory machinery that regulates the execution of different genetic and/or biochemical programs in response to specific physical or chemical signals. In the last two decades, there has been tremendous progress in our molecular understanding on how signals are detected, the allosteric mechanisms that control intramolecular information transmission and the specificity determinants that guarantee correct wiring. All this information is starting to be exploited in the development of new synthetic networks. Connecting multiple molecular players, analogous to programming lines of code, can provide the resources to build new sophisticated biocomputing systems. The Synthetic Biology field is starting to revolutionize several scientific fields, such as biomedicine and agriculture, propelling the development of new solutions. Expanding the spectrum of available nanodevices in the toolbox is key to unleash its full potential. This review aims to discuss, from a structural perspective, how to take advantage of the vast array of sensor and effector protein modules involved in two-component systems for the construction of new synthetic circuits.

Horizontal transfer of the rfb cluster in Leptospira is a genetic determinant of serovar identity.

Leptospira bacteria comprise numerous species, several of which cause serious disease to a broad range of hosts including humans. These spirochetes exhibit large intraspecific variation, resulting in complex tabulations of serogroups/serovars that crisscross the species classification. Serovar identity, linked to biological/clinical phenotypes, depends on the structure of surface-exposed LPS. Many LPS biosynthesis-encoding genes reside within the chromosomic rfb gene cluster. However, the genetic basis of intraspecies variability is not fully understood, constraining diagnostics/typing methods to cumbersome serologic procedures. We now show that the gene content of the rfb cluster strongly correlates with Leptospira serovar designation. Whole-genome sequencing of pathogenic L. noguchii, including strains of different serogroups, reveals that the rfb cluster undergoes extensive horizontal gene transfer. The rfb clusters from several Leptospira species disclose a univocal correspondence between gene composition and serovar identity. This work paves the way to genetic typing of Leptospira serovars, and to pinpointing specific genes within the distinct rfb clusters, encoding host-specific virulence traits. Further research shall unveil the molecular mechanism of rfb transfer among Leptospira strains and species.

An allosteric switch ensures efficient unidirectional information transmission by the histidine kinase DesK from Bacillus subtilis.

Phosphorylation carries chemical information in biological systems. In two-component systems (TCSs), the sensor histidine kinase and the response regulator are connected through phosphoryl transfer reactions that may be uni- or bidirectional. Directionality enables the construction of complex regulatory networks that optimize signal propagation and ensure the forward flow of information. We combined x-ray crystallography, hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, and systems-integrative kinetic modeling approaches to study phosphoryl flow through the Bacillus subtilis thermosensing TCS DesK-DesR. The allosteric regulation of the histidine kinase DesK was critical to avoid back transfer of phosphoryl groups and futile phosphorylation-dephosphorylation cycles by isolating phosphatase, autokinase, and phosphotransferase activities. Interactions between the kinase's ATP-binding domain and the regulator's receiver domain placed the regulator in two distinct positions in the phosphotransferase and phosphatase complexes, thereby determining whether a key glutamine residue in DesK was properly situated to assist in the dephosphorylation reaction. Moreover, an energetically unfavorable phosphotransferase conformation when DesK was not phosphorylated minimized reverse phosphoryl transfer. DesR dimerization and a dissociative phosphoryl transfer reaction also enforced the direction of phosphoryl flow. Shorter or longer distances between the phosphoryl acceptor and donor residues shifted the phosphoryl transfer equilibrium by modulating the stabilizing effect of the Mg2+ cofactor. These mechanisms control the directionality of signal transmission and show how structure-encoded allostery stores and transmits information in signaling systems.

Diving into the complexity of the spirochetal endoflagellum.

Spirochaetes, a phylum that includes medically important pathogens such as the causative agents of Lyme disease, syphilis, and leptospirosis, are in many ways highly unique bacteria. Their cell morphology, subcellular organization, and metabolism reveal atypical features. Spirochetal motility is also singular, dependent on the presence of periplasmic flagella or endoflagella, inserted subterminally at cell poles and not penetrating the outer membrane and elongating outside the cell as in enterobacteria. In this review we present a comprehensive comparative genomics analysis of endoflagellar systems in spirochetes, highlighting recent findings on the flagellar basal body and filament. Continued progress in understanding the function and architecture of spirochetal flagella is uncovering paradigm-shifting mechanisms of bacterial motility.

3D cryo-EM imaging of bacterial flagella: Novel structural and mechanistic insights into cell motility.

Bacterial flagella are nanomachines that enable cells to move at high speeds. Comprising 25 and more different types of proteins, the flagellum is a large supramolecular assembly organized into three widely conserved substructures: a basal body including the rotary motor, a connecting hook, and a long filament. The whole flagellum from Escherichia coli weighs ∼20 MDa, without considering its filament portion, which is by itself a ∼1.6 GDa structure arranged as a multimer of ∼30,000 flagellin protomers. Breakthroughs regarding flagellar structure and function have been achieved in the last few years, mainly because of the revolutionary improvements in 3D cryo-EM methods. This review discusses novel structures and mechanistic insights derived from such high-resolution studies, advancing our understanding of each one of the three major flagellar segments. The rotation mechanism of the motor has been unveiled with unprecedented detail, showing a two-cogwheel machine propelled by a Brownian ratchet device. In addition, by imaging the flagellin-like protomers that make up the hook in its native bent configuration, their unexpected conformational plasticity challenges the paradigm of a two-state conformational rearrangement mechanism for flagellin-fold proteins. Finally, imaging of the filaments of periplasmic flagella, which endow Spirochete bacteria with their singular motility style, uncovered a strikingly asymmetric protein sheath that coats the flagellin core, challenging the view of filaments as simple homopolymeric structures that work as freely whirling whips. Further research will shed more light on the functional details of this amazing nanomachine, but our current understanding has definitely come a long way.

Listeria innocua isolated from diseased ruminants harbour minor virulence genes of L. monocytogenes.

Listeriosis is one of the most common nervous diseases in ruminants, and is caused almost exclusively by the Gram-positive bacterium, Listeria monocytogenes. However, there are few reports of listeriosis associated with L. innocua, which is genetically closely related to L. monocytogenes, but considered non-pathogenic. In this work, we report two cases of suppurative meningoencephalitis in apparently previously healthy ruminants from different farms, in which two strains of L. innocua were recovered. The whole genomes from both isolates were sequenced, allowing phylogenetic analyses to be performed, which indicated that the two strains were very closely related. Virulence determinants were searched, especially genes coding for the main L. monocytogenes virulence factors which have been previously described in L. innocua. Surprisingly, the two isolates do not possess such virulence determinants. Instead, both strains carried a set of genes that encode for other virulence factors of the genus Listeria detected  using the Virulence Factor Database (VFDB): iap (division and invasion of host cells), lpeA (entry into non-professional phagocytes cells), fbpA (multifunctional virulence factor, including adherence to host cells), lspA (surface protein anchoring), lap (adhesion to enterocytes and trans epithelial translocation), pdgA (resistance to lysozyme), oatA (resistance to different antimicrobial compounds and also required for growth inside macrophages), lplA1 (use of host-metabolites for in vivo growth), gtcA (catalyses teichoic acid of bacterial wall), prsA2 (cell invasion, vacuole lysis and intracellular growth), clpC, clpE and clpP (survival under several stress conditions). These genes among others detected, could be involved in the ability of L. innocua to produce damage in animal and human hosts. These results highlight the multifactorial profile of Listeria pathogenesis and the need for comprehensive scientific research that address microbiological, environmental and veterinary aspects of listeriosis.

Role of the major determinant of polar flagellation FlhG in the endoflagella-containing spirochete Leptospira.

Spirochetes can be distinguished from other bacteria by their spiral-shaped morphology and subpolar periplasmic flagella. This study focused on FlhF and FlhG, which control the spatial and numerical regulation of flagella in many exoflagellated bacteria, in the spirochete Leptospira. In contrast to flhF which seems to be essential in Leptospira, we demonstrated that flhG- mutants in both the saprophyte L. biflexa and the pathogen L. interrogans were less motile than the wild-type strains in gel-like environments but not hyperflagellated as reported previously in other bacteria. Cryo-electron tomography revealed that the distance between the flagellar basal body and the tip of the cell decreased significantly in the flhG- mutant in comparison to wild-type and complemented strains. Additionally, comparative transcriptome analyses of L. biflexa flhG- and wild-type strains showed that FlhG acts as a negative regulator of transcription of some flagellar genes. We found that the L. interrogans flhG- mutant was attenuated for virulence in the hamster model. Cross-species complementation also showed that flhG is not interchangeable between species. Our results indicate that FlhF and FlhG in Leptospira contribute to governing cell motility but our data support the hypothesis that FlhF and FlhG function differently in each bacterial species, including among spirochetes.

AID overexpression leads to aggressive murine CLL and nonimmunoglobulin mutations that mirror human neoplasms.

Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease that exemplifies this process and is a model for neoplasms in general, we created transgenic mice overexpressing the enzyme activation-induced deaminase (AID), which has a normal function of inducing DNA mutations in B lymphocytes. AID not only allows normal B lymphocytes to develop more effective immunoglobulin-mediated immunity, but is also able to mutate nonimmunoglobulin genes, predisposing to cancer. In CLL, AID expression correlates with poor prognosis, suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Eµ-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in nonimmunoglobulin genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically similar amino acid substitutions as in human CLL and lymphoma. Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

The crystal structure of yeast regulatory subunit reveals key evolutionary insights into Protein Kinase A oligomerization.

Protein Kinase A (PKA) is a widespread enzyme that plays a key role in many signaling pathways from lower eukaryotes to metazoans. In mammals, the regulatory (R) subunits sequester and target the catalytic (C) subunits to proper subcellular locations. This targeting is accomplished by the dimerization and docking (D/D) domain of the R subunits. The activation of the holoenzyme depends on the binding of the second messenger cAMP. The only available structures of the D/D domain proceed from mammalian sources. Unlike dimeric mammalian counterparts, the R subunit from Saccharomyces cerevisiae (Bcy1) forms tetramers in solution. Here we describe the first high-resolution structure of a non-mammalian D/D domain. The tetramer in the crystals of the Bcy1 D/D domain is a dimer of dimers that retain the classical D/D domain fold. By using phylogenetic and structural analyses combined with site-directed mutagenesis, we found that fungal R subunits present an insertion of a single amino acid at the D/D domain that shifts the position of a downstream, conserved arginine. This residue participates in intra-dimer interactions in mammalian D/D domains, while due to this insertion it is involved in inter-dimer contacts in Bcy1, which are crucial for the stability of the tetramer. This surprising finding challenges well-established concepts regarding the oligomeric state within the PKAR protein family and provides important insights into the yet unexplored structural diversity of the D/D domains and the molecular determinants of R subunit oligomerization.

Mycobacterium tuberculosis FasR senses long fatty acyl-CoA through a tunnel and a hydrophobic transmission spine.

Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control.

Causes of abortion in dairy cows in Uruguay / Causas de aborto em bovinos de leite no Uruguai

A case series study was conducted to determine the frequency of causes of abortion in dairy cattle in Uruguay. The sample size of 102 cases was composed of 53 fetuses, 35 fetuses with placentas, and 14 placentas without an associated fetus. All cases underwent gross and microscopic pathologic examinations as well as microbiological and serological testing. The etiology was determined in 54 (53%) of cases, 51 of which were caused by infectious agents. Within the observed 102 cases, 30 (29%) were caused by Neospora caninum, six (6%) by Coxiella burnetii and two (2%) by Campylobacter fetus subsp. venerealis. Bovine Parainfluenza-3 virus and Salmonella enterica serovar Newport caused one abortion each. Opportunistic bacteria (Escherichia coli, Streptococcus sp., Staphylococcus sp., Mannheimia sp., Trueperella pyogenes, and Providencia stuartii) were associated with 11 abortions. In two cases the fetal death was attributed to dystocia, and in one case the fetus had a congenital mesothelioma. Bovine viral diarrhea virus (BVDV) infection was identified in three fetuses; two of which were co-infected with and had typical lesions of N. caninum. No lesions were observed in the other fetus infected by BVDV. Leptospira interrogans was identified in one fetus without lesions. Despite the relatively low overall success rate in establishing an etiological diagnosis in cases of abortion in cattle, a systemic workup of bovine abortion is necessary to establish prevention and control strategies. This also facilitates monitoring and surveillance of reproductive diseases in dairy cattle, some of which represent a risk to public health.(AU)

Uma série de casos foi estudada para determinar a frequência de causas do aborto em bovinos leiteiros no Uruguai. A amostra, de 102 casos, foi composta por 53 fetos, 35 fetos com placentas e 14 placentas sem feto associado. Todos os casos foram submetidos a exames patológicos macroscópicos e microscópicos, além de testes microbiológicos e sorológicos. A etiologia foi determinada em 54 (53%) dos casos, 51 dos quais foram causados por agentes infecciosos. Nos 102 casos observados, 30 (29%) foram causados por Neospora caninum, seis (6%) por Coxiella burnetii e dois (2%) por Campylobacter fetus subsp. venerealis. O vírus da Parainfluenza-3 e Salmonella enterica serovar Newport causaram um aborto cada. Bactérias oportunistas (Escherichia coli, Streptococcus sp., Staphylococcus sp., Mannheimia sp., Trueperella pyogenes e Providencia stuartii) foram associadas a 11 abortos. Em dois casos, a morte fetal foi atribuída a distocia e, em um caso, o feto apresentava mesotelioma congênito. A infecção pelo vírus da diarreia viral bovina (BVDV) foi identificada em três fetos; dois dos quais foram co-infectados e apresentavam lesões típicas de N. caninum. Não foram observadas lesões no outro feto infectado pelo BVDV. Leptospira interrogans foi identificada em um feto sem lesões. Apesar da relativamente baixa taxa de sucesso no diagnóstico etiológico nos casos de aborto em bovinos, é necessário o diagnóstico sistemático dos abortos para estabelecer estratégias de prevenção e controle. Isso também facilita o monitoramento e a vigilância de doenças reprodutivas em bovinos leiteiros, algumas das quais representam um risco para a saúde pública.(AU)

An asymmetric sheath controls flagellar supercoiling and motility in the leptospira spirochete.

Spirochete bacteria, including important pathogens, exhibit a distinctive means of swimming via undulations of the entire cell. Motility is powered by the rotation of supercoiled 'endoflagella' that wrap around the cell body, confined within the periplasmic space. To investigate the structural basis of flagellar supercoiling, which is critical for motility, we determined the structure of native flagellar filaments from the spirochete Leptospira by integrating high-resolution cryo-electron tomography and X-ray crystallography. We show that these filaments are coated by a highly asymmetric, multi-component sheath layer, contrasting with flagellin-only homopolymers previously observed in exoflagellated bacteria. Distinct sheath proteins localize to the filament inner and outer curvatures to define the supercoiling geometry, explaining a key functional attribute of this spirochete flagellum.

Protein Dynamics in Phosphoryl-Transfer Signaling Mediated by Two-Component Systems.

The ability to perceive the environment, an essential attribute in living organisms, is linked to the evolution of signaling proteins that recognize specific signals and execute predetermined responses. Such proteins constitute concerted systems that can be as simple as a unique protein, able to recognize a ligand and exert a phenotypic change, or extremely complex pathways engaging dozens of different proteins which act in coordination with feedback loops and signal modulation. To understand how cells sense their surroundings and mount specific adaptive responses, we need to decipher the molecular workings of signal recognition, internalization, transfer, and conversion into chemical changes inside the cell. Protein allostery and dynamics play a central role. Here, we review recent progress on the study of two-component systems, important signaling machineries of prokaryotes and lower eukaryotes. Such systems implicate a sensory histidine kinase and a separate response regulator protein. Both components exploit protein flexibility to effect specific conformational rearrangements, modulating protein-protein interactions, and ultimately transmitting information accurately. Recent work has revealed how histidine kinases switch between discrete functional states according to the presence or absence of the signal, shifting key amino acid positions that define their catalytic activity. In concert with the cognate response regulator's allosteric changes, the phosphoryl-transfer flow during the signaling process is exquisitely fine-tuned for proper specificity, efficiency and directionality.

Draft Genome Sequences of 40 Pathogenic Leptospira Strains Isolated from Cattle in Uruguay.

Pathogenic Leptospira species represent a major concern for livestock but also for human health, as they cause zoonotic infections. Forty strains representing L. interrogans, L. borgpetersenii, and L. noguchii were isolated from naturally infected cattle in Uruguay. Here, we report the whole-genome sequences for these strains.

Molecular adaptations of NADP-malic enzyme for its function in C4 photosynthesis in grasses.

In C4 grasses of agronomical interest, malate shuttled into the bundle sheath cells is decarboxylated mainly by nicotinamide adenine dinucleotide phosphate (NADP)-malic enzyme (C4-NADP-ME). The activity of C4-NADP-ME was optimized by natural selection to efficiently deliver CO2 to Rubisco. During its evolution from a plastidic non-photosynthetic NADP-ME, C4-NADP-ME acquired increased catalytic efficiency, tetrameric structure and pH-dependent inhibition by its substrate malate. Here, we identified specific amino acids important for these C4 adaptions based on strict differential conservation of amino acids, combined with solving the crystal structures of maize and sorghum C4-NADP-ME. Site-directed mutagenesis and structural analyses show that Q503, L544 and E339 are involved in catalytic efficiency; E339 confers pH-dependent regulation by malate, F140 is critical for the stabilization of the oligomeric structure and the N-terminal region is involved in tetramerization. Together, the identified molecular adaptations form the basis for the efficient catalysis and regulation of one of the central biochemical steps in C4 metabolism.

Two-Component Sensing and Regulation: How Do Histidine Kinases Talk with Response Regulators at the Molecular Level?

Perceiving environmental and internal information and reacting in adaptive ways are essential attributes of living organisms. Two-component systems are relevant protein machineries from prokaryotes and lower eukaryotes that enable cells to sense and process signals. Implicating sensory histidine kinases and response regulator proteins, both components take advantage of protein phosphorylation and flexibility to switch conformations in a signal-dependent way. Dozens of two-component systems act simultaneously in any given cell, challenging our understanding about the means that ensure proper connectivity. This review dives into the molecular level, attempting to summarize an emerging picture of how histidine kinases and cognate response regulators achieve required efficiency, specificity, and directionality of signaling pathways, properties that rely on protein:protein interactions. α helices that carry information through long distances, the fine combination of loose and specific kinase/regulator interactions, and malleable reaction centers built when the two components meet emerge as relevant universal principles.

Glucose-6-Phosphate Dehydrogenase from the Human Pathogen Trypanosoma cruzi Evolved Unique Structural Features to Support Efficient Product Formation.

Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme supplying reducing power (NADPH) to the cells, by oxidation of glucose-6-phosphate (G6P), and in the process providing a precursor of ribose-5-phosphate. G6PDH is also a virulence factor of pathogenic trypanosomatid parasites. To uncover the biochemical and structural features that distinguish TcG6PDH from its human homolog, we have solved and analyzed the crystal structures of the G6PDH from Trypanosoma cruzi (TcG6PDH), alone and in complex with G6P. TcG6PDH crystallized as a tetramer and enzymatic assays further indicated that the tetramer is the active form in the parasite, in contrast to human G6PDH, which displays higher activity as a dimer. This quaternary structure was shown to be particularly stable. The molecular reasons behind this disparity were unveiled by structural analyses: a TcG6PDH-specific residue, R323, is located at the dimer-dimer interface, critically contributing with two salt bridges per subunit that are absent in the human enzyme. This explains why TcG6PDH dimerization impaired enzyme activity. The parasite protein is also distinct in displaying a 37-amino-acid extension at the N-terminus, which comprises the non-conserved C8 and C34 involved in the covalent linkage of two neighboring protomers. In addition, a cysteine triad (C53, C94 and C135) specific of Kinetoplastid G6PDHs proved critical for stabilization of TcG6PDH active site. Based on the structural and biochemical data, we posit that the N-terminal region and the catalytic site are highly dynamic. The unique structural features of TcG6PDH pave the way toward the design of efficacious and highly specific anti-trypanosomal drugs.

Isolation of pathogenic Leptospira strains from naturally infected cattle in Uruguay reveals high serovar diversity, and uncovers a relevant risk for human leptospirosis.

Leptospirosis is a neglected zoonosis with worldwide distribution. The causative agents are spirochete bacteria of the Leptospira genus, displaying huge diversity of serovars, the identity of which is critical for effective diagnosis and vaccination purposes. Among many other mammalian species, Leptospira infects cattle, eliciting acute signs in calves, and chronic disease in adult animals often leading to abortions. In South America, and including in Uruguay, beef and dairy export are leading sources of national income. Despite the importance of bovine health, food safety, and bovine-related dissemination of leptospirosis to humans, extremely limited information is available as to the identity of Leptospira species and serovars infecting cattle in Uruguay and the South American subcontinent. Here we report a multicentric 3-year study resulting in the isolation and detailed characterization of 40 strains of Leptospira spp. obtained from infected cattle. Combined serologic and molecular typing identified these isolates as L. interrogans serogroup Pomona serovar Kennewicki (20 strains), L. interrogans serogroup Canicola serovar Canicola (1 strain), L. borgpetersenii serogroup Sejroe serovar Hardjo (10 strains) and L. noguchii (9 strains). The latter showed remarkable phenotypic and genetic variability, belonging to 6 distinct serogroups, including 3 that did not react with a large panel of reference serogrouping antisera. Approximately 20% of cattle sampled in the field were found to be shedding pathogenic Leptospira in their urine, uncovering a threat for public health that is being largely neglected. The two L. interrogans serovars that we isolated from cattle displayed identical genetic signatures to those of human isolates that had previously been obtained from leptospirosis patients. This report of local Leptospira strains shall improve diagnostic tools and the understanding of leptospirosis epidemiology in South America. These strains could also be used as new components within bacterin vaccines to protect against the pathogenic Leptospira strains that are actually circulating, a direct measure to reduce the risk of human leptospirosis.

Conformational plasticity of the response regulator CpxR, a key player in Gammaproteobacteria virulence and drug-resistance.

The transcriptional regulator CpxR mediates an adaptive response to envelope stress, tightly linked to virulence and antibiotics resistance in several Gammaproteobacteria pathogens. In this work, we integrated crystallographic and small-angle X-ray scattering data to gain insights into the structure and conformational plasticity of CpxR from Escherichia coli. CpxR dimerizes through two alternative interaction surfaces. Moreover, widely different CpxR conformations coexist in solution, from compact to fully extended ones. The possible functional implications of these structural features are discussed.

SIMBAD: a sequence-independent molecular-replacement pipeline.

The conventional approach to finding structurally similar search models for use in molecular replacement (MR) is to use the sequence of the target to search against those of a set of known structures. Sequence similarity often correlates with structure similarity. Given sufficient similarity, a known structure correctly positioned in the target cell by the MR process can provide an approximation to the unknown phases of the target. An alternative approach to identifying homologous structures suitable for MR is to exploit the measured data directly, comparing the lattice parameters or the experimentally derived structure-factor amplitudes with those of known structures. Here, SIMBAD, a new sequence-independent MR pipeline which implements these approaches, is presented. SIMBAD can identify cases of contaminant crystallization and other mishaps such as mistaken identity (swapped crystallization trays), as well as solving unsequenced targets and providing a brute-force approach where sequence-dependent search-model identification may be nontrivial, for example because of conformational diversity among identifiable homologues. The program implements a three-step pipeline to efficiently identify a suitable search model in a database of known structures. The first step performs a lattice-parameter search against the entire Protein Data Bank (PDB), rapidly determining whether or not a homologue exists in the same crystal form. The second step is designed to screen the target data for the presence of a crystallized contaminant, a not uncommon occurrence in macromolecular crystallography. Solving structures with MR in such cases can remain problematic for many years, since the search models, which are assumed to be similar to the structure of interest, are not necessarily related to the structures that have actually crystallized. To cater for this eventuality, SIMBAD rapidly screens the data against a database of known contaminant structures. Where the first two steps fail to yield a solution, a final step in SIMBAD can be invoked to perform a brute-force search of a nonredundant PDB database provided by the MoRDa MR software. Through early-access usage of SIMBAD, this approach has solved novel cases that have otherwise proved difficult to solve.