A bacterial surface layer protein exploits multistep crystallization for rapid self-assembly.

Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the Caulobacter crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multistep assembly pathway. This pathway involves 2 domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2-dimensional (2D) crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using a time course of electron cryo-microscopy (Cryo-EM) imaging reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the 2 domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.

A Caulobacter crescentus Microbicide Protects from Vaginal Infection with HIV-1JR-CSF in Humanized Bone Marrow-Liver-Thymus Mice.

Over 2 million people are infected with HIV-1 annually. Approximately half of these new infections occur in women residing in low-income countries, where their access to and control over HIV-1 preventative measures are often limited, indicating that female-controlled prevention options for HIV-1 are urgently needed. Microbicides that can be topically applied to the vaginal tract in advance of sexual activity represent a promising female-controlled prevention option for HIV-1. We have previously described the development of an HIV-1-specific microbicide using the surface or S-layer recombinant protein display capabilities of the nonpathogenic, freshwater bacterium Caulobacter crescentus Recombinant C. crescentus bacteria were created that displayed proteins that interfere with the HIV-1 attachment and entry process and that were able to provide significant protection of TZM-bl cells from infection with HIV-1 pseudovirus. These studies have been expanded to investigate if these recombinant C. crescentus bacteria are able to maintain efficacy with replication-competent HIV-1 and both TZM-bl cells and human peripheral blood mononuclear cells (PBMCs). In addition, we utilized the humanized bone marrow-liver-thymus (BLT) mouse model to determine if vaginal application of recombinant C. crescentus at the time of HIV-1JR-CSF infection could provide protection from HIV-1 infection. Recombinant C. crescentus bacteria expressing Griffithsin, GB virus C E2 protein, elafin, α-1-antitrypsin, indolicidin, and the fusion inhibitor T-1249 were able to protect 40 to 75% of the BLT mice from vaginal infection with HIV-1JR-CSF, with C. crescentus bacteria expressing Griffithsin being the most effective. Taken together, these data suggest that a C. crescentus-based microbicide could be a safe and effective method for HIV-1 prevention.IMPORTANCE Human immunodeficiency virus (HIV) disproportionally infects young women in sub-Saharan Africa. Current HIV-1 prevention options have had limited success among women, suggesting that alternative, female-controlled prevention options need to be developed. Microbicides that can be applied to the vaginal tract are a promising prevention option. In this study, we describe the testing of 15 potential candidates for inhibition of HIV-1 infection in a humanized mouse model of HIV-1 infection. Four of these candidates were able to provide significant protection from vaginal infection with HIV-1, with the most successful candidate protecting 75% of the mice from infection. This study describes the preclinical testing of a new strategy that could be a safe and effective option for HIV-1 prevention in women.

Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer.

Surface layers (S-layers) are paracrystalline, proteinaceous structures found in most archaea and many bacteria. Often the outermost cell envelope component, S-layers serve diverse functions including aiding pathogenicity and protecting against predators. We report that the S-layer of Caulobacter crescentus exhibits calcium-mediated structural plasticity, switching irreversibly between an amorphous aggregate state and the crystalline state. This finding invalidates the common assumption that S-layers serve only as static wall-like structures. In vitro, the Caulobacter S-layer protein, RsaA, enters the aggregate state at physiological temperatures and low divalent calcium ion concentrations. At higher concentrations, calcium ions stabilize monomeric RsaA, which can then transition to the two-dimensional crystalline state. Caulobacter requires micromolar concentrations of calcium for normal growth and development. Without an S-layer, Caulobacter is even more sensitive to changes in environmental calcium concentration. Therefore, this structurally dynamic S-layer responds to environmental conditions as an ion sensor and protects Caulobacter from calcium deficiency stress, a unique mechanism of bacterial adaptation. These findings provide a biochemical and physiological basis for RsaA's calcium-binding behavior, which extends far beyond calcium's commonly accepted role in aiding S-layer biogenesis or oligomerization and demonstrates a connection to cellular fitness.

Proposal of Henriciella barbarensis sp. nov. and Henriciella algicola sp. nov., stalked species of the genus and emendation of the genus Henriciella.

Two Gram-negative, heterotrophic, aerobic, prosthecated, marine bacteria, designated strains MCS23T and MCS27T, were isolated from seawater samples. NaCl was required for growth. The major polar lipid detected in strain MCS27T was phosphatidylglycerol, whereas those detected in MCS23T were phosphatidylglycerol, sulfoquinovosyl diacylglycerol and 1,2-diacyl-3-α-d-glucuronopyranosyl-sn-glycerol taurineamide. The most abundant cellular fatty acids were C18 : 1ω7 and C16 : 0, hydroxyl-fatty acids were 3-OH C12 : 0 in both strains and 3-OH C11 : 0 in MCS23T. Strains MCS23T and MCS27T had DNA G+C contents of 57.0 and 55.0 mol%, respectively. The two strains shared 99.3 % 16S rRNA gene sequence similarity; levels of similarity with the type strains of species of the genus Henriciella were 99.4-97.8 % but DNA-DNA hybridizations were 53 % or lower. Besides their 16S rRNA gene sequences, the novel strains can be differentiated from other species of the genus Henriciella by cell morphology, lipid and fatty acid patterns and enzyme activities. The data obtained led to the identification of two novel species, for which the names Henriciella barbarensis sp. nov. (type strain MCS23T=LMG 28705T=CCUG 66934T) and Henriciella algicola sp. nov. (type strain MCS27T=LMG 29152T=CCUG 67844T) are proposed. As these two novel species are the first prosthecate species in the genus Henriciella, an emended genus description is also provided.

Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation.

Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior. Caulobacter crescentus is unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFa and RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology to Escherichia coli TolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFa and RsaFb are not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFa and RsaFb are required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFa and RsaFb led to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFa and RsaFb led to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFa and RsaFb in cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in C. crescentusIMPORTANCE Decreased growth rate and reduced cell fitness are common side effects of protein production in overexpression systems. Inclusion bodies typically form inside the cell, largely due to a lack of sufficient export machinery to transport the overexpressed proteins to the extracellular environment. This phenomenon can conceivably also occur in natural systems. As one example of a system evolved to prevent intracellular protein accumulation, our study demonstrates that Caulobacter crescentus has two homologous outer membrane transporter proteins that are involved in S-layer export. This is an interesting case study that demonstrates how bacteria can evolve redundancy to ensure adequate protein export functionality and maintain high cellular fitness. Moreover, we provide evidence that these two outer membrane proteins, although being the closest C. crescentus homologs to TolC in E. coli, do not process TolC functionality in C. crescentus.

Transposon Mutagenesis Paired with Deep Sequencing of Caulobacter crescentus under Uranium Stress Reveals Genes Essential for Detoxification and Stress Tolerance.

UNLABELLED: The ubiquitous aquatic bacterium Caulobacter crescentus is highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of how C. crescentus tolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in the C. crescentus genome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters (rsaFa and rsaFb), a stress-responsive transcription factor (cztR), or a ppGpp synthetase/hydrolase (spoT) exhibited a significantly lower survival rate under U stress. RsaFa and RsaFb, which are homologues of TolC in Escherichia coli, have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation of rsaFa and rsaFb by 4- and 10-fold, respectively, in the presence of U. We additionally show that rsaFa mutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. Our results suggest a function for RsaFa and RsaFb in U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance in C. crescentus. IMPORTANCE: Caulobacter crescentus is an aerobic bacterium that is highly resistant to uranium (U) and has great potential to be used in U bioremediation, but its mechanisms of U resistance are poorly understood. We conducted a Tn-seq screen to identify genes specifically required for U resistance in C. crescentus. The genes that we identified have previously remained elusive using other omics approaches and thus provide significant insight into the mechanisms of U resistance by C. crescentus. In particular, we show that outer membrane transporters RsaFa and RsaFb, previously known as part of the S-layer export machinery, may confer U resistance by U efflux and/or by maintaining membrane integrity during U stress.

Two new species of the genus Callicera Panzer (Diptera: Syrphidae) from the Palaearctic Region.

Two new species of the genus Callicera Panzer are described from the Palaearctic region: C. exigua sp. nov. from the Russian Altay and C. scintilla sp. nov. from Jordan. A key is provided for all Callicera species of the Palaearctic Region.

The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex.

The ß-barrel assembly machine (BAM) complex is an essential feature of all bacteria with an outer membrane. The core subunit of the BAM complex is BamA and, in Escherichia coli, four lipoprotein subunits: BamB, BamC, BamD and BamE, also function in the BAM complex. Hidden Markov model analysis was used to comprehensively assess the distribution of subunits of the BAM lipoproteins across all subclasses of proteobacteria. A patchwork distribution was detected which is readily reconciled with the evolution of the α-, ß-, γ-, δ- and ε-proteobacteria. Our findings lead to a proposal that the ancestral BAM complex was composed of two subunits: BamA and BamD, and that BamB, BamC and BamE evolved later in a distinct sequence of events. Furthermore, in some lineages novel lipoproteins have evolved instead of the lipoproteins found in E. coli. As an example of this concept, we show that no known species of α-proteobacteria has a homologue of BamC. However, purification of the BAM complex from the model α-proteobacterium Caulobacter crescentus identified a novel subunit we refer to as BamF, which has a conserved sequence motif related to sequences found in BamC. BamF and BamD can be eluted from the BAM complex under similar conditions, mirroring the BamC:D module seen in the BAM complex of γ-proteobacteria such as E. coli.

Cauliform bacteria lacking phospholipids from an abyssal hydrothermal vent: proposal of Glycocaulis abyssi gen. nov., sp. nov., belonging to the family Hyphomonadaceae.

Cauliform bacteria are prosthecate bacteria often specialized for oligotrophic environments. A polyphasic approach, comprising 16S rRNA gene sequencing, lipid analysis and salt tolerance characterizations, was used to clarify the taxonomy of one isolate, strain MCS 33(T), obtained from above the hot water plume of a deep-sea hydrothermal vent near Vancouver island, Canada. Cells contained no detectable phospholipids or sulpholipids, but did contain 1,2-di-O-acyl-3-O-α-D-glucopyranosylglycerol, 1,2-di-O-acyl-3-O-α-D-glucopyranuronosylglycerol and the novel lipid 1,2-di-O-acyl-3-[O-α-D-glucopyranuronosyl]glycerol-6'-N-glycine. It is assumed that the various glucoronosyl lipids are replacing, at least partially, the phospholipids in their various tasks in the cell cycle. The G+C content of the genomic DNA of strain MCS 33(T) was 62.8 mol%, and Q10 was the predominant respiratory ubiquinone. The 16S rRNA gene sequence of this chemoheterotrophic, aerobic, moderately halophilic strain showed only a low similarity of 94.4% to that of Oceanicaulis alexandrii C116-18(T), and both strains also differed based on their lipids. Although the novel strain was isolated from seawater sampled near a hydrothermal vent, its optimum temperature for growth was 30 °C. The main cellular fatty acids were C18:1ω7c, C18:0 and the unknown fatty acid ECL 11.798, and the main hydroxy fatty acid was C12:0 3-OH. The strain is proposed to represent a novel species of a new genus, Glycocaulis abyssi gen. nov., sp. nov. The type strain of the type species is MCS 33(T) (=LMG 27140(T)=CCUG 62981(T)).

Prosthecate sphingomonads: proposal of Sphingomonas canadensis sp. nov.

Two stalked, aerobic, catalase- and oxidase-positive rod-shaped isolates, VKM B-1508 ( = CB 258) and FWC47(T), were analysed using a polyphasic approach. While the morphology and the 16S rRNA gene sequence of strain VKM B-1508 were 100% identical to the ones of Sphingomonas leidyi DSM 4733(T), the morphology of FWC47(T) was different, and the closest recognized species were Sphingomonas oligophenolica S213(T) ( = DSM 17107(T)) and Sphingomonas leidyi DSM 4733(T) with 97.2% and 97.0% 16S rRNA gene sequence similarity, respectively. DNA-DNA hybridization studies supported the differentiation of strain FWC47(T) from S. oligophenolica and S. leidyi. Strain FWC47(T) grew optimally at 28-30 °C, and pH 6.0-8.0. The major respiratory quinone was Q10 and the major polyamine was sym-homospermidine. The major fatty acids were C(17:1)ω6c and C(18:1)ω7c and C(15:0) 2-OH was the major 2-hydroxy fatty acid. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidyldimethylethylamine and unidentified sphingoglycolipids. The G+C content of the genomic DNA of strain FWC47(T) was 67.1 mol%. Strain FWC47(T) differed from S. leidyi by its ability to assimilate l-alanine, maltose and sucrose, by the presence of ß-galactosidase and α-chymotrypsin, and the lack of valine arylamidase and ß-glucosidase activities. Contrary to S. leidyi, FWC47(T) did not reduce nitrate and could not use fructose, acetate and N-acetyl-glusosamine. In the genus Sphingomonas, the dimorphic life cycle involving a prosthecate sessile and a flagellated swarmer cell was hitherto only known from Sphingomonas leidyi. Therefore, strain FWC47(T) represents an additional distinct prosthecate species in this genus for which the name Sphingomonas canadensis is proposed. The type strain is FWC47(T) ( =LMG 27141(T) =CCUG 62982(T)).

Review of the European Eumenes Latreille (Hymenoptera, Vespidae) using morphology and DNA barcodes, with an illustrated key to species.

The European species of the potter wasp genus Eumenes Latreille, 1802 (Vespidae, Eumeninae) are illustrated and a new illustrated key to the 13 recognised species is presented. Eumenesmediterraneusaemilianus Guiglia, 1951 is synonymised with E.papillarius (Christ, 1791) (syn. nov.), E.obscurus André, 1884 and E.andrei Dalla Torre, 1894 with E.pedunculatus (Panzer, 1799) (syn. nov.) and E.crimensis Blüthgen, 1938 with E.sareptanus André, 1884 (syn. nov.).

A new species of the genus Conosiphon Becker, 1923 and the first records of this genus for Europe (Diptera, Asilidae).

A new species of Conosiphon Becker, 1923, Conosiphonianus Álvarez Fidalgo & van den Broek, sp. nov., is described from Spain, representing the first record of this genus for Europe. It is illustrated in high-resolution photographs and the first ecological information is provided, as well as a key to all species tentatively placed in this genus.

The bacterial surface layer provides protection against antimicrobial peptides.

This report describes a previously unrecognized role for bacterial surface layers as barriers that confer protection against antimicrobial peptides. As antimicrobial peptides exist in natural environments, S-layers may provide a bacterial survival mechanism that has been selected for through evolution.

Evaluating secretion and surface attachment of SapA, an S-layer-associated metalloprotease of Caulobacter crescentus.

Caulobacter crescentus is used to display foreign peptides at high density as insertions into the surface (S)-layer protein (RsaA). Many recombinant RsaA proteins, however, are cleaved by SapA, a 71-kDa metalloprotease, suggesting a role in maintaining S-layer integrity. When overexpressed on a multicopy plasmid SapA was detected on the surface by fluorescent antibody only if RsaA and the O-side chain of LPS that mediates S-layer attachment were removed by mutation, indicating an outer membrane location beneath the S-layer. Secretion was mediated by the RsaA type 1 transporter since secretion was eliminated in transporter deficient strains or by C-terminal deletions in SapA (the presumed location of type 1 secretion signals). Secretion was required to become an active protease; mass spectrometry suggested this might be due to N-terminal processing during secretion, a feature shared with other type 1-secreted proteases. Overexpression leads to additional processing C-terminal to the protease domain, producing a 45-kDa protein. This was demonstrated to be self-processing. Deletion analysis revealed the C-terminal 100 amino acids were sufficient for anchoring and secretion. When protein G was fused to the last 238 amino acids of SapA it was secreted, surface attached and bound immunoglobulin, indicating potential for foreign protein display.

A pictorial key for identification of the hoverflies (Diptera: Syrphidae) of the Madeira Archipelago.

Background: Syrphid flies are important ecological indicators and provide crucial ecosystem services, being important pollinators and biological control agents of insect pests. These charismatic insects are conspicuous and, due to their size and colourful patterns, are relatively easy to identify. However, the lack of user-friendly literature (e.g. photographic guides) for most areas may hamper its wider selection as a study group in biodiversity and ecological studies. The syrphid fauna of Madeira Archipelago comprises 26 species, including four endemics (Eumerushispidus Smit, Aguiar & Wakeham-Dawson, 2004; Melanostomawollastoni Wakeham-Dawson, Aguiar, Smit, McCullough & Wyatt, 2004; Myathropausta, Wollaston, 1858 and Xanthandrusbabyssa, Walker, 1849), but, despite the current good taxonomic knowledge on this group, information on species distribution, ecology and conservation is still lacking. Here, we provide a pictorial key to the adult hoverflies of Madeira Archipelago highlighting diagnostic characteristics and present photographs of both males and females (in dorsal and lateral views) in colour plates. The key and plates will help researchers to differentiate these species, thus encouraging the use of this insect group in future bioindication studies. In addition, this study also aims to engage a broader audience of non-experts in improving the knowledge on the distribution and ecology of Madeira syrphids. New information: We provide a checklist for the hoverflies of Madeira Archipelago and a pictorial key to help on species identification.

A new species of the genus Psilota Meigen, 1822 (Diptera: Syrphidae) from China.

The genus Psilota Meigen, 1822 is recorded for the first time from China, and the species Psilota bashanensis Huo and Zhao sp. nov. is described and illustrated based on the adult male. The complete cytochrome c oxidase subunit I (COI) gene of this new species has been successfully obtained and compared to that of other congeneric species. An updated key to adult males of the genus Psilota from the Palaearctic Region is also provided.

Enhanced neutralization of HIV by antibodies displayed on the S-layer of Caulobacter crescentus.

Innovative methods of prevention are needed to stop the more than two million new HIV-1 infections annually, particularly in women. Local application of anti-HIV antibodies has been shown to be effective at preventing infection in nonhuman primates; however, the concentrations needed are cost prohibitive. Display of antibodies on a particulate platform will likely prolong effectiveness of these anti-HIV agents and lower the cost of goods. Here, we demonstrate that the bacterium Caulobacter crescentus and its highly expressed surface-layer (S-layer) protein can provide this antibody display platform. Caulobacters displaying protein G, alone or with CD4 codisplay, successfully captured HIV-1-specific antibodies and demonstrated functional neutralization. Compared to soluble antibodies, a neutralizing anti-HIV antibody displayed on Caulobacter was as effective or more effective at neutralizing diverse HIV-1 isolates. Moreover, when an antibody reactive with an epitope induced by CD4 binding (CD4i) was codisplayed with CD4, there was significant enhancement in HIV-1 neutralization. These results suggest that caulobacters displaying anti-HIV antibodies offer a distinct improvement in the use of antibodies as microbicides. Furthermore, these reagents can specifically evaluate anti-HIV antibodies in concert with other HIV-1 blocking agents to assess the most suitable tools for conversion to scFvs, allowing for direct display within the S-layer protein and further reducing cost of goods. In summary, C. crescentus, which can be easily produced and chemically stabilized at low cost, is well suited for engineering as an effective platform, offering an inexpensive way to produce and deliver HIV-1-specific microbicides.

The European Stag Beetle (Lucanus cervus) Monitoring Network: International Citizen Science Cooperation Reveals Regional Differences in Phenology and Temperature Response.

To address the decline in biodiversity, international cooperation in monitoring of threatened species is needed. Citizen science can play a crucial role in achieving this challenging goal, but most citizen science projects have been established at national or regional scales. Here we report on the establishment and initial findings of the European Stag Beetle Monitoring Network (ESBMN), an international network of stag beetle (Lucanus cervus) monitoring schemes using the same protocol. The network, started in 2016, currently includes 14 countries (see results) but with a strong variation in output regarding the number of transects (148 successful transects in total) and transect walks (1735). We found differences across European regions in the number of stag beetles recorded, related to phenology and temperature, but not for time of transect start. Furthermore, the initial experiences of the ESBMN regarding international cooperation, citizen science approach, and drop-out of volunteers is discussed. An international standardised protocol that allows some local variation is essential for international collaboration and data management, and analysis is best performed at the international level, whereas recruiting, training, and maintaining volunteers is best organised locally. In conclusion, we appeal for more joint international citizen science-based monitoring initiatives assisting international red-listing and conservation actions.

Analysis of the intact surface layer of Caulobacter crescentus by cryo-electron tomography.

The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.

Subtomogram alignment by adaptive Fourier coefficient thresholding.

In the past few years, three-dimensional (3D) subtomogram alignment has become an important tool in cryo-electron tomography (CET). This technique allows one to produce higher resolution images of structures which can not be reconstructed using single-particle methods. Building on previous work, we present a new dissimilarity measure between subtomograms that works well for the noisy images that often occur in CET images. A technique that is more robust to noise provides the ability to analyze macromolecules in thicker samples such as whole cells or lower the defocus in thinner samples to push the first zero of the Contrast Transfer Function (CTF). Our method, Threshold Constrained Cross-Correlation (TCCC), uses statistics of the noise to automatically select only a small percentage of the Fourier coefficients to compute the cross-correlation, which has two main advantages: first, it reduces the influence of the noise by looking at only those peaks dominated by signal; and second, it avoids the missing wedge normalization problem since we consider the same number of coefficients for all possible pairs of subtomograms. We present results with synthetic and real data to compare our approach with other existing methods under different SNR and missing wedge conditions, and show that TCCC improves alignment results for datasets with SNR<0.1. We have made our source code freely available for the community.