Tomo Live: an on-the-fly reconstruction pipeline to judge data quality for cryo-electron tomography workflows.

Data acquisition and processing for cryo-electron tomography can be a significant bottleneck for users. To simplify and streamline the cryo-ET workflow, Tomo Live, an on-the-fly solution that automates the alignment and reconstruction of tilt-series data, enabling real-time data-quality assessment, has been developed. Through the integration of Tomo Live into the data-acquisition workflow for cryo-ET, motion correction is performed directly after each of the acquired tilt angles. Immediately after the tilt-series acquisition has completed, an unattended tilt-series alignment and reconstruction into a 3D volume is performed. The results are displayed in real time in a dedicated remote web platform that runs on the microscope hardware. Through this web platform, users can review the acquired data (aligned stack and 3D volume) and several quality metrics that are obtained during the alignment and reconstruction process. These quality metrics can be used for fast feedback for subsequent acquisitions to save time. Parameters such as Alignment Accuracy, Deleted Tilts and Tilt Axis Correction Angle are visualized as graphs and can be used as filters to export only the best tomograms (raw data, reconstruction and intermediate data) for further processing. Here, the Tomo Live algorithms and workflow are described and representative results on several biological samples are presented. The Tomo Live workflow is accessible to both expert and non-expert users, making it a valuable tool for the continued advancement of structural biology, cell biology and histology.

A 10-year meta-analysis of membrane protein structural biology: Detergents, membrane mimetics, and structure determination techniques.

Structure determination of membrane proteins is critical to the molecular understanding of many life processes, yet it has historically been a technically challenging endeavor. This past decade has given rise to a number of technological advancements, techniques, and reagents, which have facilitated membrane protein structural biology, resulting in an ever-growing number of membrane protein structures determined. To collate these advances, we have mined available literature to analyze the purification and structure determination specifics for all uniquely solved membrane protein structures from 2010 to 2019. Our analyses demonstrate the strong impact of single-particle cryo-electron microscopy on the field and illustrate how this technique has affected detergent and membrane mimetic usage. Furthermore, we detail how different structure determination methods, taxonomic domains and protein classes have unique detergent/membrane mimetic profiles, highlighting the importance of tailoring their selection. Our analyses provide a quantitative overview of where the field of membrane protein structural biology stands and how it has developed over time. We anticipate that these will serve as a useful tool to streamline future membrane protein structure determination by guiding the choice of detergent/membrane mimetic.

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24.

Ste24 enzymes, a family of eukaryotic integral membrane proteins, are zinc metalloproteases (ZMPs) originally characterized as "CAAX proteases" targeting prenylated substrates, including a-factor mating pheromone in yeast and prelamin A in humans. Recently, Ste24 was shown to also cleave nonprenylated substrates. Reduced activity of the human ortholog, HsSte24, is linked to multiple disease states (laminopathies), including progerias and lipid disorders. Ste24 possesses a unique "α-barrel" structure consisting of seven transmembrane (TM) α-helices encircling a large intramembranous cavity (~14 000 Å3 ). The catalytic zinc, coordinated via a HExxH…E/H motif characteristic of gluzincin ZMPs, is positioned at one of the cavity's bases. The interrelationship between Ste24 as a gluzincin, a long-studied class of soluble ZMPs, and as a novel cavity-containing integral membrane protein protease has been minimally explored to date. Informed by homology to well-characterized soluble, gluzincin ZMPs, we develop a model of Ste24 that provides a conceptual framework for this enzyme family, suitable for development and interpretation of structure/function studies. The model consists of an interfacial, zinc-containing "ZMP Core" module surrounded by a "ZMP Accessory" module, both capped by a TM helical "α-barrel" module of as yet unknown function. Multiple sequence alignment of 58 Ste24 orthologs revealed 38 absolutely conserved residues, apportioned unequally among the ZMP Core (18), ZMP Accessory (13), and α-barrel (7) modules. This Tripartite Architecture representation of Ste24 provides a unified image of this enzyme family.

A critical evaluation of in silico methods for detection of membrane protein intrinsic disorder.

Intrinsically disordered regions in proteins possess important biological roles including transcriptional regulation, molecular recognition, and provision of sites for posttranslational modification. In three-dimensional crystallization of both soluble and membrane proteins, identification and removal of disordered regions is often necessary for obtaining crystals possessing sufficient long-range order for structure determination. Disordered regions can be identified experimentally, with techniques such as limited proteolysis coupled with mass spectrometry, or computationally, by using disorder prediction programs, of which many are available. Although these programs use various methods to predict disorder from a protein's primary sequence, they all were developed using information derived from soluble protein structures. Therefore, their performance and accuracy when applied to integral membrane proteins remained an open question. We evaluated the performance of 13 disorder prediction programs on a dataset containing 343 membrane proteins, and upon subdatasets containing only α-helical or ß-barrel proteins. These programs were ranked using multiple metrics, including metrics specifically created for membrane proteins. Analysis of these data shows a clear distinction between programs that accurately predict disordered regions in membrane proteins and programs which perform poorly, and allows for the robust integration of in silico disorder prediction into our PSI:Biology membrane protein structural genomics pipeline.

Structural and dynamic studies of the transcription factor ERG reveal DNA binding is allosterically autoinhibited.

The Ets-Related Gene (ERG) belongs to the Ets family of transcription factors and is critically important for maintenance of the hematopoietic stem cell population. A chromosomal translocation observed in the majority of human prostate cancers leads to the aberrant overexpression of ERG. We have identified regions flanking the ERG Ets domain responsible for autoinhibition of DNA binding and solved crystal structures of uninhibited, autoinhibited, and DNA-bound ERG. NMR-based measurements of backbone dynamics show that uninhibited ERG undergoes substantial dynamics on the millisecond-to-microsecond timescale but autoinhibited and DNA-bound ERG do not. We propose a mechanism whereby the allosteric basis of ERG autoinhibition is mediated predominantly by the regulation of Ets-domain dynamics with only modest structural changes.

Structure of the integral membrane protein CAAX protease Ste24p.

Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a-factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.

Crystallization of Pseudomonas aeruginosa AmrZ protein: development of a comprehensive method for obtaining and optimization of protein-DNA crystals.

The AmrZ protein from the pathogenic bacterium Pseudomonas aeruginosa is a transcription factor that activates and represses the genes for several potent virulence factors, which gives the bacteria a selective advantage in infection. AmrZ was crystallized in complex with DNA containing the amrZ1 repressor binding site. Obtaining crystals of the complex required the integration of a number of well known techniques along with the development of new methods. Here, these processes are organized and combined into a comprehensive method which yielded diffraction-quality crystals. Part of this method included thorough data mining of the crystallization conditions of protein-DNA complexes to create a new directed crystallization screen. An optimized technique for the verification of protein-DNA complexes in crystals is also presented. Taken together, the methods described in this article attempt to streamline the difficult process of obtaining diffraction-quality crystals of protein-DNA complexes through the organization of older methods combined with the introduction of new techniques.

The transcription factor AmrZ utilizes multiple DNA binding modes to recognize activator and repressor sequences of Pseudomonas aeruginosa virulence genes.

AmrZ, a member of the Ribbon-Helix-Helix family of DNA binding proteins, functions as both a transcriptional activator and repressor of multiple genes encoding Pseudomonas aeruginosa virulence factors. The expression of these virulence factors leads to chronic and sustained infections associated with worsening prognosis. In this study, we present the X-ray crystal structure of AmrZ in complex with DNA containing the repressor site, amrZ1. Binding of AmrZ to this site leads to auto-repression. AmrZ binds this DNA sequence as a dimer-of-dimers, and makes specific base contacts to two half sites, separated by a five base pair linker region. Analysis of the linker region shows a narrowing of the minor groove, causing significant distortions. AmrZ binding assays utilizing sequences containing variations in this linker region reveals that secondary structure of the DNA, conferred by the sequence of this region, is an important determinant in binding affinity. The results from these experiments allow for the creation of a model where both intrinsic structure of the DNA and specific nucleotide recognition are absolutely necessary for binding of the protein. We also examined AmrZ binding to the algD promoter, which results in activation of the alginate exopolysaccharide biosynthetic operon, and found the protein utilizes different interactions with this site. Finally, we tested the in vivo effects of this differential binding by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence and show that differences in binding alone do not affect transcriptional regulation.

AmrZ beta-sheet residues are essential for DNA binding and transcriptional control of Pseudomonas aeruginosa virulence genes.

AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the ß-sheet for DNA binding, while the α-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a ß-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ ß-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection.

Cooperative DNA binding and communication across the dimer interface in the TREX2 3' --> 5'-exonuclease.

The activity of human TREX2-catalyzed 3' --> 5'-deoxyribonuclease has been analyzed in steady-state and single turnover kinetic assays and in equilibrium DNA binding studies. These kinetic data provide evidence for cooperative DNA binding within TREX2 and for coordinated catalysis between the TREX2 active sites supporting a model for communication between the protomers of a TREX2 dimer. Mobile loops positioned adjacent to the active sites provide the major DNA binding contribution and facilitate subsequent binding into the active sites. Mutations of three arginine residues on these loops cause decreased TREX2 activities by up to 60-fold. Steady-state kinetic assays of these arginine to alanine TREX2 variants result in increased K(m) values for DNA substrate with no effect on k(cat) values indicating contributions exclusively to DNA binding by all three of the loop arginines. TREX2 heterodimers were prepared to determine whether exonuclease activity in one protomer is communicated to the opposing protomer. Evidence for communication across the dimer interface is provided by the 7-fold lower catalytic activity measured in the TREX2(WT/H188A) heterodimer compared with the TREX2(WT) homodimer, contrasting the 2-fold lower activity measured in the TREX2(WT/R163A,R165A,R167A) heterodimer. The measured activity in TREX2(WT/H188A) heterodimer indicates that defective catalysis in one protomer reduces activity in the opposing protomer. A DNA binding analysis of TREX2 and the heterodimers indicates a cooperative binding effect within the TREX2 protomer. Finally, single turnover kinetic assays identify DNA binding as the rate-limiting step in TREX2 catalysis.