Biology subjects

Otwinowski, Z.

Publications and source records attributed to Otwinowski, Z..

9 recordsLinked to original sources

bioRxiv2026

Cryo-EM structure of detergent-bound Vibrio cholerae MakA captures a candidate intermediate between the soluble form and mature assembly of an α-pore-forming toxin

The tripartite toxin MakA/MakB/MakE from Vibrio cholerae belongs to a family of -pore-forming toxins that undergo extensive conformational rearrangements upon interacting with membrane. While soluble structures of MakA, MakB and MakE have been determined, the structural basis of membrane association and pore assembly remains poorly understood. Here, we report a cryo-electron microscopy structure of a detergent-associated form of MakA at a global resolution of 3.13 Angstrom, with focused refinement of a flexible region improving the local resolution to 2.68 Angstrom. This structure consists of a trimer of MakA asymmetric homodimers, in which each subunit adopts a transmembrane -helical conformation distinct from the previously reported soluble structure. The asymmetric homodimer closely resembles the building block observed in the previously reported helical MakA assembly and is structurally similar to the homodimeric building blocks of the Aeromonas hydrophila AhlB and Serratia marcescens SmhA pores, and the heterodimeric building blocks of the Xenorhabdus nematophila XaxAB and Yersinia enterocolitica YaxAB pores, suggesting a conserved assembly principle among distantly related -pore-forming toxins. Complementary hydropathy analysis, AlphaFold2 modeling, and liposome-based assays reveal distinct roles for the three components, with MakA acting as the primary membrane-inserting subunit, MakE exhibiting low but consistent membrane-association, and MakB alone remaining largely soluble. Together, these results support a model in which the MakA asymmetric homodimer represents a plausible early membrane-insertion unit and building block for pore assembly, while the trimer of dimers represents an early assembly intermediate.

microbiology↗

bioRxiv2026

Analysis of the heterogenous structural states of the hexameric ATPase PilU of the Type IV pili from Vibrio cholerae

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 digerent combinations of open and closed interfaces even in the absence of nucleotide. Comparison with PilT highlights structural features that likely underlie PilUs weak ATPase activity and its dependence on PilT for function. Together, these findings provide a structural framework for understanding PilUs role within the T4P retraction machinery.

molecular biology↗

bioRxiv2025

Preserving Information and Integrity in Cryo-EM: A Fourier-Space Deposition Approach

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 reconstructions 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. HighlightsO_LIReciprocal-space deposition to preserve signal and uncertainty estimates. C_LIO_LIMandatory molecular masks deposition for accurate and unbiased FSC validation. C_LIO_LILimitations of half-map-based Fourier Shell Coefficients. C_LIO_LIThe difference maps can be calculated. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/690836v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1a35884org.highwire.dtl.DTLVardef@1086af4org.highwire.dtl.DTLVardef@1a5f118org.highwire.dtl.DTLVardef@177815a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Validation of 3D cryoEM single particle reconstruction correctness and handedness with Ewald's sphere correction

The correct description of quantum scattering places the observed scattering contributions on the Ewalds sphere and its Friedel mate copy. In electron microscopy, due to the large radius of the Ewalds 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 Ewalds sphere affects the overall signal-to-noise ratio.

biophysics↗

bioRxiv2024

Structural architecture of TolQ-TolR inner membrane protein complex from opportunistic pathogen Acinetobacter baumannii

Gram-negative bacteria harness the proton motive force (PMF) within their inner membrane (IM) to uphold the integrity of their cell envelope, 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-EM 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 trans-membrane funnel leading towards a cytoplasmic chamber. In contrast, the TolQ-TolR complex assumes a proton non-permeable stance, characterized by the TolQ pentamers 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. TeaserApo TolQ and TolQ-TolR structures depict structural rearrangements required for cell envelope organization in bacterial cell division.

molecular biology↗

bioRxiv2022

Monomer and dimer structures of cytochrome bo3 ubiquinol oxidase from Escherichia coli

The E. 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 on this protein, 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 [A] and 3.46 [A], respectively. We have discovered that the protein can form a dimer in 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 each monomer, except the movement of a loop in subunit IV (residues 67-74).

biophysics↗

bioRxiv2022

CryoEM single particle reconstruction with a complex-valued particle stack

Single particle reconstruction (SPR) in cryoEM is an image processing task with an elaborate hierarchy that starts with a large number of 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 an exit-wave-reconstruction-based image, in which CTF correction is implicitly applied as a real component of the image. The final CTF correction that we later refine and apply 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 while calculations crucial for high resolution reconstruction such as Ewald sphere correction, aberration refinement, and particlespecific defocus refinement can be performed on the small box data. Highlights{blacksquare} A complex-valued particle stack facilitates flexible and fast data processing {blacksquare}A real-valued, compact particle stack can be used at intermediate steps {blacksquare}Whole micrograph padding is used from the beginning of data analysis

biophysics↗

bioRxiv2020

The His-tag as a decoy modulating preferred orientation in cryoEM

The His-tag is a widely used affinity tag that facilitates purification by means of affinity chromatography of recombinant proteins for functional and structural studies. We show here that His-tag presence affects how coproheme decarboxylase interacts with the water-air interface during grid preparation for cryoEM. Depending on His-tag presence or absence, we observe significant changes in patterns of preferred orientation. The analysis of particle orientations suggests that His-tag presence can mask the hydrophobic patches on a proteins surface that mediate the interactions with the water-air interface, while the hydrophobic linker between a His-tag and the coding sequence of the protein may enhance other interactions with water-air interface. Our observations suggest that tagging, including rational design of the linkers between an affinity tag and a protein of interest, offer a promising approach to modulating interactions with the water-air interface. SynopsisA His-tag affects the interactions of particles with the water-air interface in cryo-electron microscopy (cryoEM) single particle reconstruction (SPR), and thus may be used to modulate these interactions, including inducing changes in patterns of preferred orientation.

biophysics↗

bioRxiv2019

High-resolution cryo-EM reconstructions in the presence of substantial aberrations

The beam-image shift method accelerates data acquisition in cryo-EM single particle reconstruction (cryo-EM SPR) by fast repositioning of the imaging area, but at the cost of more severe and complex optical aberrations.\n\nWe analyze here how uncorrected anti-symmetric aberrations, such as coma and trefoil, affect cryo-EM SPR results, and then infer an analytical formula quantifying information loss due to their presence that explains why Fourier-shell coefficient (FSC)-based statistics may report significantly overestimated resolution if these aberrations are not fully corrected. We validate our analysis with reference-based aberration refinement for two cryo-EM SPR datasets acquired with a 200 kV microscope in the presence of coma exceeding 40 {micro}m, and obtained 2.3 and 2.7 [A] reconstructions for 144 and 173 kDa particles, respectively.\n\nOur results provide a description of an efficient approach for assessing information loss in cryo-EM SPR data acquired in the presence of higher-order aberrations and address inconsistent guidelines regarding the level of aberrations acceptable in cryo-EM SPR experiments.

biophysics↗