Many bacteria, and most archaea, have a paracrystalline protein surface layer (S-layer) encapsulating their outer membrane or cell wall. For pathogenic Rickettsia species, an S-layer has been hypothesized to be comprised of the outer membrane protein OmpB. We used cryo-electron tomography (cryoET) to image the Rickettsia parkeri cell surface and observed a repetitive S-layer structure proximal to the outer membrane of the bacterium that is absent in an ompB STOP ::tn mutant. Our data reveal that OmpB is essential for the Rickettsia S-layer and suggest that we can leverage cryoET to examine rickettsial S-layer structure and function.

The chlamydial Major Outer Membrane Protein (MOMP) is a promising subunit vaccine candidate due to its abundance in the outer membrane (OM), the presence of four surface-exposed variable domains (VDs) containing neutralizing and serotyping epitopes, and confirmed T-cell epitopes located in its constant domains (CDs). However, recombinant and denatured forms of MOMP have failed to elicit immune responses comparable to native preparations, indicating that conformation is critical for immunogenicity. Here, we present two cryo-EM structures of native Chlamydia muridarum MOMP, isolated from infectious elementary bodies (EBs). EB MOMP forms a distinct trimer with a stem of three narrow, non-permeable β-barrels and an extracellular, folded antigenic cap that displays VDs. In complex with a neutralizing Fab fragment of a conformational antibody, the cap undergoes structural reorganization that shows how epitope presentation is modulated by immune engagement. These structures reveal the molecular basis of MOMP's serovar specificity and provide a template for structure-based vaccine design.

Type IV pili (T4P) mediate surface motility, host interactions, and DNA uptake through cycles of extension and retraction. While the primary retraction ATPase PilT has been extensively characterized, its homolog PilU remains less well understood despite being demonstrated as a PilT-dependent retraction ATPase. Here, we determined six PilU structures by cryo-electron microscopy and x-ray crystallography. The structures reveal a homohexameric assembly stabilized by interactions between the C-terminal catalytic domain of one subunit and the N-terminal PAS-like domain of a neighboring subunit. PilU adopts multiple conformational states, exhibiting different combinations of open and closed interfaces even in the absence of nucleotide. Comparison with PilT highlights structural features that likely underlie PilU's weak ATPase activity and its dependence on PilT for function. Together, these findings provide a structural framework for understanding PilU's role within the T4P retraction machinery.

The correct description of quantum scattering places the observed scattering contributions on the Ewald's sphere and its Friedel mate copy. In electron microscopy, due to the large radius of the Ewald's sphere, these scattering contributions are typically merged during data analysis. We present an approach that separates and factorizes those contributions into real and imaginary components of the image. When an inverted solution is calculated, the map derived from the real component of the image generates an inverted solution, while the map derived from the imaginary component of the image generates an inverted and sign-flipped solution. Therefore, the sign of correlation between reconstructions derived from the real and imaginary components provides the automatic determination of handedness and additional validation for the quality of 3D reconstructions. The factorization and its implementation are robust enough to be routinely used in single-particle reconstructions, even at resolutions below the limit where the curvature of the Ewald's sphere affects the overall signal-to-noise ratio.

In cryoEM 3D reconstruction, noise is spread over the entire 3D space, while the signal is present only within the molecular mask. The signal-to-noise ratio (SNR) within the molecular mask is the SNR of interest in data analysis. Any method that does not apply the molecular mask to noise underestimates the SNR. For instance, Fourier Shell Correlation (FSC) curves recalculated between two half-maps after the data are deposited in the PDB do not apply the molecular mask to noise, consequently providing a misleading impression of resolution. These approaches, relying on calculations without mask constraints on noise, try to avoid introducing biases within the mask area that could potentially arise at low resolution. Here, we present a method of FSC calculations in which masking does not introduce spurious correlation between two maps. In this approach, we split the particle image signal in real space into two components: 1) the real part, which is the signal used in traditional calculations; and 2) the imaginary part, which is the signal present only when Ewald's sphere calculation is considered. If the two components are used in reconstruction separately, they are independent of each other. Then, the FSC is calculated between two maps derived from the real part of all particles for one map and from the imaginary part of all particles for the second map. Typically, reconstruction from the real component is used to refine particle orientations, introducing bias into subsequent reconstruction based on the real component. However, reconstruction based on the imaginary component is not biased by the refinement using only the real component. This way, reconstruction from the imaginary component can be used as an unbiased reference for validating reconstruction from the real component. This validation works even at relatively low resolution (10 Å data if the number of particles is large), despite relying on Ewald's sphere curvature. The byproduct of this validation is the determination of the structure's handedness, which only requires medium resolution data. In cryoEM 3D reconstruction, noise is spread over the entire 3D space, while the signal is present only within the molecular mask. The signal-to-noise ratio (SNR) within the molecular mask is the SNR of interest in data analysis. Any method that does not apply the molecular mask to noise underestimates the SNR. For instance, Fourier Shell Correlation (FSC) curves recalculated between two half-maps after the data are deposited in the PDB do not apply the molecular mask to noise, consequently providing a misleading impression of resolution. These approaches, relying on calculations without mask constraints on noise, try to avoid introducing biases within the mask area that could potentially arise at low resolution. Here, we present a method of FSC calculations in which masking does not introduce spurious correlation between two maps. In this approach, we split the particle image signal in real space into two components: 1) the real part, which is the signal used in traditional calculations; and 2) the imaginary part, which is the signal present only when Ewald's sphere calculation is considered. If the two components are used in reconstruction separately, they are independent of each other. Then, the FSC is calculated between two maps derived from the real part of all particles for one map and from the imaginary part of all particles for the second map. Typically, reconstruction from the real component is used to refine particle orientations, introducing bias into subsequent reconstruction based on the real component. However, reconstruction based on the imaginary component is not biased by the refinement using only the real component. This way, reconstruction from the imaginary component can be used as an unbiased reference for validating reconstruction from the real component. This validation works even at relatively low resolution (10 Å data if the number of particles is large), despite relying on Ewald's sphere curvature. The byproduct of this validation is the determination of the structure's handedness, which only requires medium resolution data.

ABSTRACT During infection, bacterial pathogens rely on secreted virulence factors to manipulate the host cell. However, in gram-positive bacteria, the molecular mechanisms underlying the folding and activity of these virulence factors after membrane translocation are not clear. Here, we solved the protein structures of two secreted parvulin and two secreted cyclophilin-like peptidyl-prolyl isomerase (PPIase) ATP-independent chaperones found in gram-positive streptococcal species. The extracellular parvulin-type PPIase, PrsA in Streptococcus pneumoniae and Streptococcus mutans maintain dimeric crystal structures reminiscent of folding catalysts that consist of two domains, a PPIase and foldase domain. Structural comparison of the two cyclophilin-like extracellular chaperones from S. pneumoniae and Streptococcus pyogenes with other cyclophilins demonstrates that this group of cyclophilin-like chaperones has novel structural appendages formed by 9- and 24-residue insertions. Furthermore, we demonstrate that deletion of prsA and slrA genes impairs the secretion of the cholesterol-dependent pore-forming toxin, pneumolysin in S. pneumoniae. Using protein pull-down and biophysical assays, we demonstrate a direct interaction between PrsA and SlrA with Ply. Then, we developed chaperone-assisted folding assays that show that the S. pneumoniae PrsA and SlrA extracellular chaperones accelerate pneumolysin folding. In addition, we demonstrate that SlrA and, for the first time, S. pyogenes PpiA exhibit PPIase activity and can bind the immunosuppressive drug, cyclosporine A. Altogether, these findings suggest a mechanistic role for streptococcal PPIase chaperones in the activity and folding of secreted virulence factors such as pneumolysin.IMPORTANCEStreptococcal species are a leading cause of lower respiratory infections that annually affect millions of people worldwide. During infection, streptococcal species secrete a medley of virulence factors that allow the bacteria to colonize and translocate to deeper tissues. In many gram-positive bacteria, virulence factors are secreted from the cytosol across the bacterial membrane in an unfolded state. The bacterial membrane-cell wall interface is exposed to the potentially harsh extracellular environment, making it difficult for native virulence factors to fold before being released into the host. ATP-independent PPIase-type chaperones, PrsA and SlrA, are thought to facilitate folding and stabilization of several unfolded proteins to promote the colonization and spread of streptococci. Here, we present crystal structures of the molecular chaperones of PrsA and SlrA homologs from streptococcal species. We provide evidence that the Streptococcus pyogenes SlrA homolog, PpiA, has PPIase activity and binds to cyclosporine A. In addition, we show that Streptococcus pneumoniae PrsA and SlrA directly interact and fold the cholesterol-dependent pore-forming toxin and critical virulence determinant, pneumolysin.

Current guidelines for depositing cryogenic electron microscopy single particle reconstruction (cryo-EM SPR) data require submission of unfiltered, unmasked, and unsharpened raw half-maps. The Fourier Shell Correlation (FSC) between the half-maps is then used as a proxy for the signal-to-noise ratio (SNR) to estimate the reconstruction's resolution. This policy was introduced to enable independent validation of reported resolutions. Although developed to safeguard data integrity and minimize bias, these guidelines do not account for specific features of modern cryo-EM processing software, in particular weighting schemes that are not retained in half-map depositions and yet in general are necessary to recapitulate resolution estimates. As a results, resolution estimates and other validation statistics based on half-maps FSC may be under- or overestimated. Here, we describe the limitations of the current deposition guidelines and propose an alternative: depositing cryo-EM results in Fourier (reciprocal) space together with the mandatory deposit of molecular masks or their descriptors. This approach addresses the current limitations, preserves critical information from the reconstruction process, and better supports downstream analyses.

Ultra-high-resolution crystal structures of proteins provide critical insights into protein structure, dynamics, hydrogen bonding, and solvent networks. Crambin, a small hydrophobic storage protein consisting of 46 residues (4.7 kDa), is found in the embryonic tissue of seeds from Crambe abyssinica. This protein is renowned for its ability to crystallize readily, forming some of the best-ordered macromolecular crystals known, which diffract X-rays to the highest sub-atomic resolution recorded for any protein to date.We have previously reported the room temperature structure of crambin, refined to an exceptional resolution of 0.70 Å using SHELXL. That analysis revealed intricate details of the dynamic solvent network, characterized by alternative side chain conformations and shifts in water molecule positions. In this work, we extend our investigation by presenting new structural data collected at cryogenic temperatures: 15K using liquid helium and 100K using liquid nitrogen cooling.We will report the ultra-high-resolution structures at 15K and 100K, providing a comparative analysis of the solvent networks across these different temperature datasets. This comparison aims to deepen our understanding of the solvent and protein dynamics, offering valuable insights into the protein interactions within solvent environments. Our findings underscore the significance of ultrahigh-resolution crystallography in elucidating the complex interplay between proteins and their solvent environments, with potential implications for the broader field of structural biology.

Prokaryotic heme biosynthesis in Gram-positive bacteria follows the coproporphyrin-dependent heme biosynthesis pathway. The last step in this pathway is catalyzed by the enzyme coproheme decarboxylase, which oxidatively transforms two propionate groups into vinyl groups yielding heme b. The catalytic reaction cycle of coproheme decarboxylases exhibits four different states: the apo-form, the substrate (coproheme)-bound form, a transient three-propionate intermediate form (monovinyl, monopropionate deuteroheme; MMD), and the product (heme b)-bound form. In this study, we used cryogenic electron microscopy single-particle reconstruction (cryo-EM SPR) to characterize structurally the apo and heme b-bound forms of actinobacterial coproheme decarboxylase from Corynebacterium diphtheriae. The flexible loop that connects the N-terminal and the C-terminal ferredoxin domains of coproheme decarboxylases plays an important role in interactions between the enzyme and porphyrin molecule. To understand the role of this flexible loop, we performed molecular dynamics simulations on the apo and heme b coproheme decarboxylase from Corynebacterium diphtheriae. Our results are discussed in the context of the published structural information on coproheme-bound and MMD-bound coproheme decarboxylase and with respect to the reaction mechanism. Having structural information of all four enzymatically relevant states helps in understanding structural restraints with a functional impact.

Polymyxins are last-resort antimicrobial peptides administered clinically against multi-drug resistant bacteria, specifically in the case of Gram-negative species. However, an increasing number of these pathogens employ a defense strategy that involves a relay of enzymes encoded by the pmrE ( ugd ) loci and the arnBCDTEF operon. The pathway modifies the lipid-A component of the outer membrane (OM) lipopolysaccharide (LPS) by adding a 4-amino-4-deoxy- l -arabinose ( L -Ara4N) headgroup, which renders polymyxins ineffective. Here, we report the cryo-EM SPR structures of glycosyltransferase ArnC from Salmonella typhimurium determined in apo and UDP-bound forms at resolutions 2.75 Å and 3.8 Å, respectively. The structure of the ArnC protomer comprises three distinct regions: an N-terminal glycosyltransferase domain, transmembrane region, and the interface helices (IHs). ArnC forms a tetramer with C2 symmetry, where the C-terminal strand inserts into the adjacent protomer. This tetrameric state is further stabilized by two distinct interfaces formed by ArnC that form a network of hydrogen bonds and salt bridges. The binding of UDP induces conformational changes that stabilize the loop between residues H201 to S213, and part of the putative catalytic pocket formed by IH1 and IH2. The surface property analysis revealed a hydrophobic cavity formed by TM1 and TM2 in the apo state, which is disrupted upon UDP binding. The comparison of ArnC structures to their homologs GtrB and DPMS suggests the key residues involved in ArnC catalytic activity.

Gram-negative bacteria harness the proton motive force (PMF) within their inner membrane (IM) to uphold cell envelope integrity, an indispensable aspect for both division and survival. The IM TolQ-TolR complex is the essential part of the Tol-Pal system, serving as a conduit for PMF energy transfer to the outer membrane. Here we present cryo-electron microscopy reconstructions of Acinetobacter baumannii TolQ in apo and TolR-bound forms at atomic resolution. The apo TolQ configuration manifests as a symmetric pentameric pore, featuring a transmembrane funnel leading toward a cytoplasmic chamber. In contrast, the TolQ-TolR complex assumes a proton nonpermeable stance, characterized by the TolQ pentamer's flexure to accommodate the TolR dimer, where two protomers undergo a translation-based relationship. Our structure-guided analysis and simulations support the rotor-stator mechanism of action, wherein the rotation of the TolQ pentamer harmonizes with the TolR protomers' interplay. These findings broaden our mechanistic comprehension of molecular stator units empowering critical functions within the Gram-negative bacterial cell envelope.

The Mediator complex facilitates interactions between transcription factors and RNA polymerase II, a process that is required for host gene transcription, including in response to viral infections. Among the many subunits in the Mediator complex, the MED25 subunit has been shown to be a target for viral activators during infection. Here we provide the molecular basis for the interaction between human respiratory syncytial virus (hRSV) nonstructural 1 protein (NS1) and the activator interaction domain (ACID) of MED25. The X-ray crystal structure of the complex revealed that NS1 straddles and binds two faces of MED25 ACID. This interaction is distinct from previously known viral activators. Importantly, our data support the conformational flexibility of viral transcriptional regulators. Furthermore, ChIP-seq and RNA-seq analysis identified the ATF3 transcription factor and a role for NS1/Mediator/ATF3 interaction in host gene regulation in hRSV infections. Our findings provide a molecular basis for hRSV NS1-based regulation of host gene transcription and reveal how viruses exploit the conformational heterogeneity at fuzzy transcription activator interfaces.

Ultrahigh-resolution structures provide unprecedented details about protein dynamics, hydrogen bonding and solvent networks. The reported 0.70 Å, room-temperature crystal structure of crambin is the highest-resolution ambient-temperature structure of a protein achieved to date. Sufficient data were collected to enable unrestrained refinement of the protein and associated solvent networks using SHELXL. Dynamic solvent networks resulting from alternative side-chain conformations and shifts in water positions are revealed, demonstrating that polypeptide flexibility and formation of clathrate-type structures at hydrophobic surfaces are the key features endowing crambin crystals with extraordinary diffraction power.

TolQ-TolR-TolA is an energy-transducing inner-membrane sub-complex conserved across Gram-negative bacteria. It maintains connection with periplasmic TolB-Pal sub-complex in an energy-dependent manner through TolA protein spanning the periplasm. Utilizing proton motive force (PMF), Tol-Pal complex contributes to the integrity of outer membrane and the whole cell envelope. TolQ remains the only part of Tol-Pal complex with unknown structure up to now. To address this, we determined 3.02 and 3.34 Å cryo-electron microscopy (cryo-EM) structures of the integral inner-membrane protein TolQ and in complex with TolR, respectively from Acinetobacter baumannii.

Single particle reconstruction (SPR) in cryoEM is an image processing task with an elaborate hierarchy that starts with many very noisy multi-frame images. Efficient representation of the intermediary image structures is critical for keeping the calculations manageable. One such intermediary structure is called a particle stack and contains cut-out images of particles in square boxes of predefined size. The micrograph that is the source of the boxed images is usually corrected for motion between frames prior to particle stack creation. However, the contrast transfer function (CTF) or its Fourier Transform point spread function (PSF) are not considered at this step. Historically, the particle stack was intended for large particles and for a tighter PSF, which is characteristic of lower resolution data. The field now performs analyses of smaller particles and to higher resolution, and these conditions result in a broader PSF that requires larger padding and slower calculations to integrate information for each particle. Consequently, the approach to handling structures such as the particle stack should be reexamined to optimize data processing. Here we propose to use as a source image for the particle stack a complex-valued image, in which CTF correction is implicitly applied as a real component of the image. We can achieve it by applying an initial CTF correction to the entire micrograph first and perform box cutouts as a subsequent step. The final CTF correction that we refine and apply later has a very narrow PSF, and so cutting out particles from micrographs that were approximately corrected for CTF does not require extended buffering, i.e. the boxes during the analysis only have to be large enough to encompass the particle. The Fourier Transform of an exit-wave reconstruction creates an image that has complex values. This is a complex value image considered in real space, opposed to standard SPR data processing where complex numbers appear only in Fourier space. This extension of the micrograph concept provides multiple advantages because the particle box size can be small and calculations crucial for high resolution reconstruction such as Ewald sphere correction, aberration refinement, and particle-specific defocus refinement can be performed on the small box data.

The Escherichia coli cytochrome bo3 ubiquinol oxidase is a four-subunit heme-copper oxidase that serves as a proton pump in the E. coli aerobic respiratory chain. Despite many mechanistic studies, it is unclear whether this ubiquinol oxidase functions as a monomer, or as a dimer in a manner similar to its eukaryotic counterparts-the mitochondrial electron transport complexes. In this study, we determined the monomeric and dimeric structures of the E. coli cytochrome bo3 ubiquinol oxidase reconstituted in amphipol by cryogenic electron microscopy single particle reconstruction (cryo-EM SPR) to a resolution of 3.15 and 3.46 Å, respectively. We have discovered that the protein can form a dimer with C2 symmetry, with the dimerization interface maintained by interactions between the subunit II of one monomer and the subunit IV of the other monomer. Moreover, the dimerization does not induce significant structural changes in the monomers, except the movement of a loop in subunit IV (residues 67-74).