Cryo Electron Microscopy

While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300 nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET. In this work, we present a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures. These coating steps eliminate the adverse effects of sample charging on imaging with the Volta phase plate, allowing data acquisition with improved contrast. We discuss optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas that make large areas of unperturbed HeLa and Chlamydomonas cells accessible for cryo-ET at molecular resolution.

Propelled by improvements in hardware for data collection and processing, single particle cryo-electron microscopy has rapidly gained relevance in structural biology. Yet, finding the conditions to stabilize a macromolecular target for imaging remains the most critical barrier to determining its structure. Attaining the optimal specimen requires the evaluation of multiple grids in a microscope as conditions are varied. While automation has significantly increased the speed of data collection, optimization is still carried out manually. This laborious process which is highly dependent on subjective assessments, inefficient and prone to error, often determines the success of a project. Here, we present SmartScope, the first framework to streamline, standardize, and automate specimen evaluation in cryo-electron microscopy. SmartScope employs deep-learningbased object detection to identify and classify features suitable for imaging, allowing it to perform thorough specimen screening in a fully automated manner. A web interface provides remote control over the automated operation of the microscope in real time and access to images and annotation tools. Manual annotations can be used to re-train the feature recognition models, leading to improvements in performance. Our automated tool for systematic evaluation of specimens streamlines structure determination and lowers the barrier of adoption for cryo-electron microscopy.

Plant membrane transporters are essential for perceiving salinity stress, transducing signals, and mitigating stress during salt exposure. These proteins reside in the membrane and are modulated by various cellular components, including membrane lipids. Certain specific lipids are known to modulate the activity of plant membrane transporters, influencing their structure, function, and distribution within the membrane. Most of these regulatory lipids are low in abundance and target specific subsets of membrane transporters based on their localization. The interaction between plant membrane transporters and lipids in response to salinity stress is a relatively underexplored area. While there are only a few instances where the roles of lipids have been directly linked to transporters during salinity stress, this review comprehensively covers salinity stress-related plant membrane transporters known to be regulated by lipids. Further, it also examines the effects of lipids and their role in regulating the major transporters present in both the plasma membrane and intracellular membranes. The lipid regulation mechanisms of the transporters are inferred from recently published structures of lipid-bound plant membrane transporters. Finally, the review discusses traditional and novel methods for identifying lipid-membrane protein interactions. This work highlights the significance of studying the membrane protein-lipid interactome in light of recent insights into lipid function and the role of membrane transporters in plant salinity tolerance.

Cryo-electron microscopy (cryo-EM) is a powerful tool for macromolecular to near-atomic resolution structure determination in the biological sciences. The specimen is maintained in a near-native environment within a thin film of vitreous ice and imaged in a transmission electron microscope. The images can then be processed by a number of computational methods to produce three-dimensional information. Recent advances in sample preparation, imaging, and data processing have led to tremendous growth in the field of cryo-EM by providing higher resolution structures and the ability to investigate macromolecules within the context of the cell. Here, we review developments in sample preparation methods and substrates, detectors, phase plates, and cryo-correlative light and electron microscopy that have contributed to this expansion. We also have included specific biological applications.

Electron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adapt...

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Wprowadzenie 1. Projektowanie nowych leków przeciwgruźliczych ukierunkowane na mykobaktyny 2. Mimetyki acyloadenylanu 2.1. Synteza analogów adenozyny modyfikowanych w pozycji 5' grupą aminoacylosulfamoilową 3. Badanie zależności pomiędzy aktywnością a modyfikacją w obrębie domeny cukrowej, arylowej oraz nukleozasadowej 4. Synteza konformacyjnie usztywnionych pochodnych Sal-AMS 5. Analogi mające na celu polepszenie parametrów farmakokinetycznych Sal-AMS Podsumowanie Piśmiennictwo cytowane Dr Katarzyna Kulik ukończyła Wydział Chemii Uniwersytetu Łódzkiego w 2005 roku i w tym samym roku rozpoczęła pracę w Zakładzie Chemii Bioorganicznej CBMM PAN w Łodzi. Stopień naukowy doktora uzyskała w 2013 roku. Jej zainteresowania naukowe dotyczą głównie projektowania, syntezy i badanie wybranych właściwości biologicznych modyfikowanych nukleozydów o potencjalnym działaniu przeciwnowotworowym i przeciwwirusowym. Dr hab. Janina Baraniak po ukończeniu studiów na Wydziale Chemii Politechniki Łódzkiej rozpoczęła pracę w CBMM PAN w Łodzi pod kierunkiem Prof. Wojciecha Steca, gdzie w 1986 roku uzyskała stopień naukowy doktora a w 2005 roku stopień doktora habilitowanego. Głównym przedmiotem zainteresowań naukowych jest chemia modyfikowanych nukleozydów, nukleotydów i oligonukleotydów. Prowadzone badania są skoncentrowane nad syntezą związków o potencjalnych właściwościach terapeutycznych jak i istotnym znaczeniu poznawczym nakierowanym na badania mechanistyczne procesów biochemicznych. Jest promotorem dwóch prac doktorskich i współautorką 68 publikacji. Od 2006 roku pracuje w Akademii im. Jana Długosza w Częstochowie.

Viruses-like particles (VLPs) are frequently being used as platforms for polyvalent display of foreign epitopes of interest on their capsid surface to improve their presentation enhancing the antigenicity and host immune response. In the present study, we used the VLPs of Tomato bushy stunt virus (TBSV), an icosahedral plant virus, as a platform to display 180 copies of 16 amino acid epitopes of ricin toxin fused to the C-terminal end of a modified TBSV capsid protein (NΔ52). Expression of the chimeric recombinant protein in insect cells resulted in spontaneous assembly of VLPs displaying the ricin epitope. Cryo-electron microscopy and image reconstruction of the chimeric VLPs at 22 Å resolution revealed the locations and orientation of the ricin epitope exposed on the TBSV capsid surface. Furthermore, injection of chimeric VLPs into mice generated antisera that detected the native ricin toxin. The ease of fusing of short peptides of 15-20 residues and their ability to form two kinds (T = 1, T = 3) of bio-nanoparticles that result in the display of 60 or 180 copies of less constrained and highly exposed antigenic epitopes makes TBSV an attractive and versatile display platform for vaccine design.

Model-based, three-dimensional reconstruction techniques depend on reliable starting models. We present an extension of the random-model method (RMM) that allows the ab initio generation of suitable starting models directly from un-averaged, experimental images of asymmetric or symmetric particles. Therefore, the asymmetric RMM can also be used to determine point-group symmetry. The procedure is facilitated by the use of a) variable angular step-sizes during iterative origin and orientation searches, b) high numbers of particle images, and c) highly defocused images. The method is inhibited by mixed-handedness orientation assignments and by particles with inconspicuous features. For symmetric particles, symmetric RMMs can overcome these deficiencies.

Correction of electron beam-induced sample motion is one of the major factors contributing to the recent resolution breakthroughs in cryo-electron microscopy. Improving the accuracy and efficiency of motion correction can lead to further resolution improvement. Based on observations that the electron beam induces doming of the thin vitreous ice layer, we developed an algorithm to correct anisotropic image motion at the single pixel level across the whole frame, suitable for both single particle and tomographic images. Iterative, patch-based motion detection is combined with spatial and temporal constraints and dose weighting. The multi-GPU accelerated program, MotionCor2, is sufficiently fast to keep up with automated data collection. The result is an exceptionally robust strategy that can work on a wide range of data sets, including those very close to focus or with very short integration times, obviating the need for particle polishing. Application significantly improves Thon ring quality and 3D reconstruction resolution. . In recent years single-particle cryo-electron microscopy (cryo-EM) has made groundbreaking advancements in obtaining atomic resolution structures of macromolecules that are either refractory to crystallization or difficult to be crystallized in specific functional states . Central to this success has been the broad application of direct electron detection cameras, especially the Gatan K2, which not only have significantly improved detective quantum efficiency (DQE) at all frequencies but also have a high output frame rate, typically 10 to 40 frames per second. Nowadays, cryo-EM images of frozen hydrated biological samples are recorded as dosefractionated stacks of sub-frames (movies) , which enable correction of beam-induced sample motion that blurs the captured images . Combining high sensitivity imaging, motion correction and advanced image processing and 3D classification strategies have made it practical to routinely determine three-dimensional (3D) reconstructions at near atomic resolution by single particle cryo-EM even for the most challenging samples 9-11 . Sample illumination with the high-energy electron beam breaks bonds, releases radiolysis products and builds up charge within the thin frozen hydrated biological samples during image recording. The result is a combination of physical and optical distortions that can significantly deteriorate sample high-resolution information through image blurring. This has been one of the major factors limiting the achievable resolution of single particle cryo-EM 12 . The concept of recording the image as movie to correct sample motion was proposed long ago , but only became practical after direct electron detection cameras were made available 7 . The fast sample motions can be measured by tracking the movement of images captured in a series of snapshots, either as the whole frame or as individual particles. Image motion can then be corrected by registering identical features in the sub-frames to each other, followed by summing the registered sub-frames to produce a motion-corrected image 6 . From the earliest studies it has been clear that the apparent sample motions can be anisotropic and idiosyncratic, such that the pattern of motion varies across the image. While in principle tracking should be straightforward, the practical challenge is the extremely low signal-to-noise ratio (SNR) in each individual sub-frame. Except for very largest particles, accurate motion measurement generally requires correlating the motion between sub-frames over large areas. That said, even sub-optimal motion correction can significantly restore high-resolution signals and improve the resolution of final 3D reconstructions 6,8 . .

Sickle cell disease (SCD) poses a significant public health challenge in Uganda, affecting approximately 1.6 million individuals and contributing to high morbidity and mortality rates. Current SCD management is often fragmented, with care primarily concentrated in specialized centers, leading to disparities in access and delayed interventions. The integration of SCD care into the primary healthcare (PHC) system has been proposed as a strategy to address these challenges. This narrative review explores the current gaps in SCD care within Uganda's healthcare system and examines the potential benefits of integrating SCD management into PHC settings to improve outcomes. A comprehensive literature review was conducted to assess existing SCD management practices, barriers within PHC systems, and outcomes from integration pilot programs. Data on healthcare provider confidence, access to diagnostic tools, and patient outcomes were analyzed to understand the current landscape and opportunities for improvement. Only 35% of healthcare providers in PHC settings reported confidence in managing SCD patients (OR: 0.45, 95% CI: 0.35-0.55). Limited access to diagnostic tools and inadequate resources further hinders timely diagnosis and treatment, contributing to preventable complications and higher healthcare costs. However, a pilot program integrating SCD care into PHC clinics demonstrated a 50% reduction in hospital admissions for pain crises among participants (OR: 2.0, 95% CI: 1.5-2.7). Integrating SCD care into Uganda's PHC framework offers significant potential to enhance early diagnosis, improve treatment adherence, and reduce hospital admissions. Coordinated care approaches could alleviate the disease burden, improve patient outcomes, and lower healthcare costs.

Virus-like particles (VLPs) derived from nonenveloped viruses result from the self-assembly of capsid proteins (CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self-assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N-and C-terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant-based production of nucleic acid-free VLPs. Remarkably, expression of N-or C-terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

At the initial stage of the cryo-electron microcopy (cryo-EM) samples irradiated by electrons, the cryo-EM samples suffer from a rapid “burst” phase (first 3~4 e−/Å2) of beam induced motion (BIM) which is too fast to be corrected by existing motion correction software, and lowers the quality of the initial frames. Therefore, these least radiation damaged, but ruined frames are commonly excluded or down-weighted during data processing, which reduces the undamaged signals in the reconstruction and decreases the reconstruction resolution by weakening the averaging power. Here, we show that increasing the freezing temperature of cryo-EM samples suppresses the BIM in this phase. The quality of initial frames is partially recovered after BIM correction and is better than that of subsequent frames in certain frames. Incorporating these initial frames into the reconstruction increases the resolution, at an equivalent of ~60% extra data. Moreover, these frames are least radiation damaged, th...

method, vs 13.2% using the rhodanine and 17.5% using the orcein method. In group B, Timm's method was positive in 40.1% while rhodanine and orcein showed positivity in 26.7%. In group C, the Timm's method stained 58.6%, rhodanine 36.6% and orcein 29.3% positively. Conclusions: Our data show that: (1) Timm's silver stain is the most sensitive method for the demonstration of copper in all cases of WD; (2) rhodanine and orcein have minor value in the diagnosis of WD, especially in the early stages of the disease; (3) to increase the diagnostic value of histochemistry for copper multiple histochemical stains in serial sections are required; and (4) although hepatic copper concentration is highest in the early stages of WD, the histochemical demonstration fails in a large number of cases.

Nudaurelia capensis  virus (NωV) is a eukaryotic RNA virus that is well suited for the study of virus maturation. The virus initially assembles at pH 7.6 into a marginally stable 480-Å procapsid formed by 240 copies of a single type of protein subunit. During maturation, which occurs during apoptosis at pH 5.0, electrostatic forces guide subunit trajectories into a robust 410-Å virion that is buttressed by subunit associated molecular switches. We discuss the competing factors in the virus capsid of requiring near-reversible interactions during initial assembly to avoid kinetic traps, while requiring robust stability to survive in the extra-cellular environment. In addition, viruses have a variety of mechanisms to deliver the genome, which must remain off while still inside the infected cell, yet turn on under the proper conditions of infection. We conclude that maturation is the process that provides a solution to these conflicting requirements through a program that is encoded in the procapsid and that leads to stability and infectivity.

A new era has begun for single particle cryo-electron microscopy (cryoEM) which can now compete with X-ray crystallography for determination of protein structures. The development of direct detectors constitutes a revolution that has led to a wave of near-atomic resolution cryoEM reconstructions. However, regardless of the sample studied, virtually all high-resolution reconstructions reported to date have been achieved using high-end microscopes. We demonstrate that the new generation of direct detectors coupled to a widely used mid-range electron microscope also enables obtaining cryoEM maps of sufficient quality for de novo modeling of protein structures of different sizes and symmetries. We provide an outline of the strategy used to achieve a 3.7 Å resolution reconstruction of Nudaurelia capensis ω virus and a 4.2 Å resolution reconstruction of the Thermoplasma acidophilum T20S proteasome. Single-particle cryo-electron microscopy (cryoEM) is currently undergoing a revolution due to the recent development of a new generation of detectors using the complementary metaloxide semiconductors technology (Kuhlbrandt, 2014). These cameras directly detect incoming electrons, without the need for a scintillator converting the electrons into photons, and are characterized by improved detective quantum efficiencies at all spatial frequencies compared to traditional charge-coupled device cameras or photographic films . The fast read-out rate of these devices also allows for recording movies composed of multiple frames during typical image exposure times enabling correction for beam-induced sample motion and stage drift to reduce image blurring .

Nudaurelia capensis w virus (NωV) is a eukaryotic RNA virus that is well suited for the study of virus maturation. The virus initially assembles at pH 7.6 into a marginally stable 480-Å procapsid formed by 240 copies of a single type of protein subunit. During maturation, which occurs during apoptosis at pH 5.0, electrostatic forces guide subunit trajectories into a robust 410-Å virion that is buttressed by subunit associated molecular switches. We discuss the competing factors in the virus capsid of requiring near-reversible interactions during initial assembly to avoid kinetic traps, while requiring robust stability to survive in the extra-cellular environment. In addition, viruses have a variety of mechanisms to deliver the genome, which must remain off while still inside the infected cell, yet turn on under the proper conditions of infection. We conclude that maturation is the process that provides a solution to these conflicting requirements through a program that is encoded in...

The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane sensor, RstB and its cognate response regulator (RR) RstA. The RstA of Klebsiella pneumoniae (kpRstA) consists of an N-terminal receiver domain (RD, residues 1-119) and a C-terminal DNA-binding domain (DBD, residues 130-236). Phosphorylation of kpRstA induces dimerization, which allows two kpRstA DBDs to bind to a tandem repeat, called the RstA box, and regulate the expression of downstream genes. Here we report the solution and crystal structures of the free kpRstA RD, DBD and DBD/RstA box DNA complex. The structure of the kpRstA DBD/RstA box complex suggests that the two protomers interact with the RstA box in an asymmetric fashion. Equilibrium binding studies further reveal that the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner. Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the ...

The AAV2.7m8 vector is an engineered capsid with a 10-amino acid insertion in Adeno-associated virus (AAV) surface variable region VIII (VR-VIII) resulting in the alteration of an antigenic region of AAV2 and the ability to efficiently transduce retina cells following intravitreal administration. Directed evolution and in vivo screening in the mouse retina isolated this vector. In the present study, we sought to identify the structural differences between a recombinant AAV2.7m8 (rAAV2.7m8) vector packaging a GFP genome and its parental serotype, AAV2, by cryo-electron microscopy (cryo-EM) and image reconstruction. The structures of rAAV2.7m8 and AAV2 were determined to 2.91 and 3.02 Å resolution, respectively. The rAAV2.7m8 amino acid side-chains for residues 219-745 (the last C-terminal residue) were interpretable in the density map with the exception of the 10 inserted amino acids. While observable in a low sigma threshold density, side-chains were only resolved at the base of the insertion, likely due to flexibility at the top of the loop. A comparison to parental AAV2 (ordered from residues 217-735) showed the structures to be similar, except at some side-chains that had different orientations and, in VR-VIII containing the 10 amino acid insertion. VR-VIII is part of an AAV2 antigenic epitope, and the *

The AAV2.7m8 vector is an engineered capsid with a 10-amino acid insertion in Adeno-associated virus (AAV) surface variable region VIII (VR-VIII) resulting in the alteration of an antigenic region of AAV2 and the ability to efficiently transduce retina cells following intravitreal administration. Directed evolution and in vivo screening in the mouse retina isolated this vector. In the present study, we sought to identify the structural differences between a recombinant AAV2.7m8 (rAAV2.7m8) vector packaging a GFP genome and its parental serotype, AAV2, by cryo-electron microscopy (cryo-EM) and image reconstruction. The structures of rAAV2.7m8 and AAV2 were determined to 2.91 and 3.02 Å resolution, respectively. The rAAV2.7m8 amino acid side-chains for residues 219-745 (the last C-terminal residue) were interpretable in the density map with the exception of the 10 inserted amino acids. While observable in a low sigma threshold density, side-chains were only resolved at the base of the insertion, likely due to flexibility at the top of the loop. A comparison to parental AAV2 (ordered from residues 217-735) showed the structures to be similar, except at some side-chains that had different orientations and, in VR-VIII containing the 10 amino acid insertion. VR-VIII is part of an AAV2 antigenic epitope, and the *

The availability of both EM and high-resolution crystallographic data for several membrane proteins (MPs) permits a detailed evaluation of the ability of molecular modeling techniques to complement EM data in the development of models of MPs. A protocol for this purpose is presented, consisting of (1) identifying transmembrane (TM) domains from sequence; (2) assigning buried and lipid-exposed faces of the TM domains; and (3) assembling the TM domains into a bundle, based on geometric restraints obtained from the EM data. The protocol is validated by predicting the structures of several 7-and 12-TM MPs to within 3-5 A ˚r.m.s.d. from their crystal structures. The protocol is applied to generate a model of the oxalate transporter OxlT, for which a high-resolution structure is not yet available.

The capsid protein (CP) of Sesbania mosaic virus (SeMV, a T ¼ 3 plant virus) consists of a disordered Nterminal R-domain and an ordered S-domain. Removal of the R-domain results in the formation of T ¼ 1 particles. In the current study, the R-domain was replaced with unrelated polypeptides of similar lengths: the B-domain of Staphylococcus aureus SpA, and SeMV encoded polypeptides P8 and P10. The chimeric proteins contained T¼ 3 or larger virus-like particles (VLPs) and could not be crystallized. The presence of metal ions during purification resulted in a large number of heterogeneous nucleoprotein complexes. NΔ65-B (R domain replaced with B domain) could also be purified in a dimeric form. Its crystal structure revealed T ¼1 particles devoid of metal ions and the B-domain was disordered. However, the B-domain was functional in NΔ65-B VLPs, suggesting possible biotechnological applications. These studies illustrate the importance of N-terminal residues, metal ions and robustness of the assembly process.

Nitric oxide (NO) deficiency plays a crucial role in the pathophysiology of sickle cell anemia (SCA), contributing to vascular dysfunction and related complications that significantly impact patient health. NO is vital for maintaining endothelial function, regulating vascular tone, and preventing platelet aggregation. In SCA, reduced NO bioavailability results from hemolysis, oxidative stress, and impaired nitric oxide synthase (NOS) activity. These factors lead to vascular issues such as pulmonary hypertension, vaso-occlusive crises, and stroke. This review explores the mechanisms behind NO depletion in SCA and emphasizes how NO deficiency worsens vascular dysfunction. The reduction of NO in SCA is mainly due to the release of cell-free hemoglobin from lysed red blood cells, which scavenges NO, along with increased production of reactive oxygen species (ROS) that break down NO. Additionally, endothelial dysfunction further decreases NO synthesis, creating a vicious cycle of declining NO availability. This causes elevated vascular resistance, impaired blood flow, and a higher risk of thrombotic events, all contributing to the morbidity and mortality observed in SCA patients.

We present a model to calculate the displacement and extension of deformable cellular cargo pulled by molecular motors stepping along cytoskeletal filaments. We consider the case of a single type of molecular motor and cytoskeletal filaments oriented in one dimension in opposite directions on either side of a cargo. We model a deformable cargo as a simple elastic spring. We simulate this tug-of-war simple exclusion process model using a Monte Carlo Gillespie algorithm and calculate the displacement and extension of the cargo for different configurations of motors and filaments. We apply our model to kinesin-1 motors on microtubules to investigate whether they are strong enough to translocate and deform the largest cellular cargo, the nucleus. We show that the extension caused by motors on a single microtubule saturates for larger numbers of motors but that the extension and displacement scales linearly with the number of microtubules. We also show how the binding and unbinding behaviors of molecular motors on microtubule filaments affect the nuclear deformation. Our modelling results correspond to experiments on cells treated with the drug kinesore, which is thought to increase rescue events resulting in more stable microtubules and more active kinesin-1 molecular motors bound to them. Both the experiments and our simulations result in nuclear deformation.

I show here that Viral Capsids can be easily destructed by means of a specific violet radiation. This possibility is extremely advantageous from the technical viewpoint, because can be very efficient to destruct viral capsids at the air, on the skin or on the oral mucosa, for example.

In recent work with large high symmetry viruses, single particle electron cryomicroscopy (cryoEM) has reached the milestone of determining near atomic resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains extraordinarily challenging. Using a newly developed single electron counting detector, we confirm that electron beam induced motion significantly degrades resolution and, importantly, show how the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy. Thus, intrinsic image information can be restored to high resolution (Thon rings visible to ~3 Å). Using this approach we determined a 3.3 Å resolution structure of a ~700 kDa protein with D7 symmetry showing clear side chain density. Our method greatly enhances image quality and data acquisition efficiency -key bottlenecks in applying near atomic resolution cryoEM to a broad range of protein samples. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The amplitude contrast of frozen-hydrated biological samples was measured using the bacterial flagellar filament embedded in vitreous ice at an accelerating voltage of 300 kV. From the mean radial amplitude spectra of overfocused images, amplitude contrast was estimated to be 6.9 ± 1.9% and 2.7 ± 1.0% of the whole contrast at the low spatial frequency range with and without energy filtering, respectively, and that of the carbon film to be 9.5 ± 2.0% and 5.8 ± 1.8%. Energy filtering effectively doubled the signal-to-noise ratio in the images of frozen-hydrated filaments, and substantially improved intensity data statistics of layer lines up to at least $25 A ˚resolution in their Fourier transforms. It also markedly improved inter-particle fitting phase residuals of averaged data at resolutions up to $15 A ˚. Using the energy filtered data recorded on a new high-performance, lens-coupled CCD camera the three-dimensional map of the flagellar filament was calculated at 8 A ˚by applying the amplitude contrast of 6.9%. The map and its mean radial density distribution validated the obtained value of the amplitude contrast.

Single particle reconstruction using the random conical tilt data collection geometry is a robust method for the initial determination of macromolecular structures by electron microscopy. Unfortunately, the broad adoption of this powerful approach has been limited by the practical challenges inherent in manual data collection of the required pairs of matching high and low tilt images (typically 60°and 0°). The microscopist is obliged to keep the imaging area centered during tilting as well as to maintain accurate focus in the tilted image while minimizing the overall electron dose, a challenging and time consuming process. To help solve these problems, we have developed an automated system for the rapid acquisition of accurately aligned and focused tilt pairs. The system has been designed to minimize the dose incurred during alignment and focusing, making it useful in both negative stain and cryo-electron microscopy. The system includes a feature for montaging untilted images to ensure that all of the particles in the tilted image may be used in the reconstruction.

Dual-axis electron microscopic tomography minimizes the missing wedge-induced resolution loss by taking two complementary tilt data sets of the same target along two orthogonal axes. The potential of this powerful approach has been hampered by the practical challenges inherent in finding the original targets that are dramatically displaced due to non-eucentric specimen rotation. Not only is the manual search for the original targets time consuming and tedious but the added dose during manual searching is uncontrollable. We have developed a hierarchical alignment scheme that allows tomographic data to be collected from an arbitrary number of target sites in one grid orientation and then to find and collect orthogonal data sets with little or no user intervention. Inspired by the successful multi-scale mapping in Leginon, our alignment is performed in three levels to gradually pinpoint the original targets. At the lowest level the grid lattice is used to determine the rotation angle and translational shift resulting from specimen rotation via auto-and cross-correlative analysis of a pair of atlas maps constructed before and after specimen rotation. The target locations are further refined at the next level using a pair of smaller atlas maps. The final refinement of target positions is done by aligning the target contained image tiles. Given the batch processing nature of this hierarchical alignment, multiple targets are initially selected in a group and then sequentially acquired. Upon completion of the data collection on all the targets along the first axis and after specimen rotation, the hierarchical alignment is performed to relocate the original targets. The data collection is then resumed on these targets for the second axis. Therefore, only one specimen rotation is needed for collecting multiple dual-axis tomographic data sets. The experiment of acquiring 20S Proteasomes dual-axis tomographic data sets in vitreous ice at 86000x CCD magnification on our FEI Tecnai Polara TF30 electron microscope has suggested that the developed scheme is very robust. The extra doses for finding and centering the original targets are almost negligible. This scheme has been integrated into UCSF Tomography software suite that can be downloaded at www.msg.ucsf.edu/tomography free for academic use.

Peer review information Nature Methods thanks David Haselbach, Mark Herzik Jr., Ellen Zhong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Arunima Singh, in collaboration with the Nature Methods team. Reprints and permissions information is available at www.nature.com/reprints.

Cryo-EM samples prepared using traditional methods often suffer from too few particles, poor particle distribution, strongly biased orientation, or damage from the air-water interface. Here we report that functionalization of graphene oxide (GO) coated grids with amino groups concentrates samples on the grid with improved distribution and orientation. By introducing a PEG spacer, particles are kept away from both the GO surface and the air-water interface, protecting them from potential denaturation.

dendrymer Rys. 1.1. Przykładowe struktury wybranych związków makroacyklicznych i makropolicyklicznych [2] Układy tego typu naleŜą do świata chemii supramolekularnej, zainicjowanej przez Jean-Marie Lehn'a [3], który wraz z Pedersenem i Cramem w 1987 roku otrzymał Nagrodę Nobla w dziedzinie chemii za osiągnięcia w zakresie chemii supramolekularnej, w tym zsyntezowanie sztucznych makromolekuł do modelowania reakcji w Ŝywych organizmach [4]. Chemia supramolekularna, zwana równieŜ chemią pozacząsteczkową [5], jest nauką o słabych oddziaływaniach, występujących w duŜej ilości róŜnorodnych układów, od prostych, takich jak wiązania wodorowe między cząsteczkami wody, do złoŜonych mechanizmów fotochemicznych i skomplikowanych procesów biologicznych, w których te siły odgrywają kluczową rolę w rozpoznawaniu molekularnym i katalizie. Oddziaływania międzycząsteczkowe stanowią podstawę procesów rozpoznawania molekularnego, reakcji, transportu, regulacji, itp., występujących w biologii, np. wiązanie substratu do białka, tworzenie kompleksów białko-białko, asocjacja antygen-antyciało, odczyt międzycząsteczkowy, translacja i transkrypcja kodu genetycznego, indukcja sygnału przez neurotransmitery, rozpoznanie komórkowe [6-12].

Finding the conditions to stabilize a macromolecular target for imaging remains the most critical barrier to determining its structure by cryo-electron microscopy (cryo-EM). While automation has significantly increased the speed of data collection, specimens are still screened manually, a laborious and subjective task that often determines the success of a project. Here, we present SmartScope, the first framework to streamline, standardize, and automate specimen evaluation in cryo-EM. SmartScope employs deep-learning-based object detection to identify and classify features suitable for imaging, allowing it to perform thorough specimen screening in a fully automated manner. A web interface provides remote control over the automated operation of the microscope in real time and access to images and annotation tools. Manual annotations can be used to re-train the feature recognition models, leading to improvements in performance. Our automated tool for systematic evaluation of specimens streamlines structure determination and lowers the barrier of adoption for cryo-EM.

Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unstained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used negative staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of negative staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor.

Przedstawiono charakterystykę oddziaływań między białkami i polisacharydami, które wynikają ze specyficznej budowy tych biopolimerów. Omówiono wymagania dotyczące substratów oraz scharakteryzowano warunki, jakie powinny zostać spełnione, aby możliwe było powstawanie kompleksów między nimi. Określono ponadto parametry występowania termodynamicznej niekompatybilności roztworów białek i polisacharydów. W dalszej części pracy podano praktyczne implikacje, jakie potencjalnie wynikają z oddziaływań tych makrocząsteczek. Opisano również konkretne przykłady zastosowań przemysłowych, wskazując przy tym na to, że oprócz aplikacji tradycyjnych w przemyśle spożywczym coraz częściej pojawiają się nowe niespożywcze zastosowania w obszarach biotechnologii, ochrony środowiska, kosmetyce i technologii materiałów biodegradowalnych.

are distorted on account of the packing in the crystal (Penczek et al., 1999). The best-defined 3D motifs, of c/o DESY Notkestraße 85 double-stranded A form rRNA, are 17-20 A ˚in diameter with major grooves that can reach 13.5 A ˚in depth. Thus, D-22603 Hamburg Germany resolutions in the 13 to 10 A ˚range are sufficient for detection and interpretation of such structural features Institute of Crystallography Russian Academy of Sciences and elements richly presented on the surface and at the subunit interface (Ban et al., 1999; Cate et al., 1999; Leninsky pr. 59 117333 Moscow Clemons et al., 1999). Here, we present an 11.5 A ˚cryo-EM map of the E. Russia coli 70S ribosome initiation-like complex with fMet-tRNA f Met at the P site. Guided by recent placements of proteins and rRNA elements in X-ray maps, we were Summary able to identify some of the ribosomal components and the nature of connections between the ribosomal sub-Over 73,000 projections of the E. coli ribosome bound units. The map also elucidates the architecture of funcwith formyl-methionyl initiator tRNA f Met were used to tionally important domains. Structural details seen at obtain an 11.5 A ˚cryo-electron microscopy map of the this resolution, especially when put into the context of complex. This map allows identification of RNA helicryo-EM mappings of EF-G (Agrawal et al., 1998, 1999a, ces, peripheral proteins, and intersubunit bridges. 2000), begin to reveal the identity and functional inter-Comparison of double-stranded RNA regions and poplay of the connections between the regions associated sitions of proteins identified in both cryo-EM and X-ray with decoding, peptidyl transfer, and GTPase activity. maps indicates good overall agreement but points to rearrangements of ribosomal components required Results for the subunit association. Fitting of known components of the 50S stalk base region into the map defines Cryo-Electron Microscopy and Three-Dimensional the architecture of the GTPase-associated center and Reconstruction reveals a major change in the orientation of the The cryo-EM density map was obtained from a total of ␣-sarcin-ricin loop. Analysis of the bridging connec-73,523 projections of the fMet-tRNA f Met •ribosome comtions between the subunits provides insight into the plex in a defocus range of 0.73 to 4.34 m (see Experidynamic signaling mechanism between the ribosomal mental Procedures). The Fourier shell correlation curve subunits. (Figure 1) of the final, contrast-transfer function-corrected reconstruction indicates a resolution of 11.5 A

In translation, elongation factor Tu (EF-Tu) molecules deliver aminoacyl-tRNAs to the mRNA-programmed ribosome. The GTPase activity of EF-Tu is triggered by ribosome-induced conformational changes of the factor that play a pivotal role in the selection of the cognate aminoacyl-tRNAs. We present a 6.7-Å cryo-electron microscopy map of the aminoacyl-tRNA·EF-Tu·GDP·kirromycin-bound Escherichia coli ribosome, together with an atomic model of the complex obtained through molecular dynamics flexible fitting. The model reveals the conformational changes in the conserved GTPase switch regions of EF-Tu that trigger hydrolysis of GTP, along with key interactions, including those between the sarcin-ricin loop and the P loop of EF-Tu, and between the effector loop of EF-Tu and a conserved region of the 16S rRNA. Our data suggest that GTP hydrolysis on EF-Tu is controlled through a hydrophobic gate mechanism.

Wykaz stosowanych skrótów Wprowadzenie 1. Alditole jako składniki strukturalne aktywnych biologicznie związków 2. Zastosowanie alditoli w syntezie polimerów 3. Metody syntezy alditoli oraz anhydroalditoli Uwagi końcowe Podziękowania Piśmiennictwo cytowane Dr Barbara Dmochowska w roku 1994 ukończyła studia chemiczne na Wydziale Chemii Uniwersytetu Gdańskiego w Gdańsku. Jest adiunktem w Pracowni Chemii Cukrów na Wydziale Chemii UG. Tematyka badawcza: tworzenie czwartorzędowych soli N-d-glikoamoniowych i alditoliloamoniowych z udziałem amin o potencjalnych właściwościach biologicznych.

With the rapid improvement of cryo-electron microscopy (cryo-EM) resolution, new computational tools are needed to assist and improve upon atomic model building and refinement options. This communication demonstrates that microscopists can now collaborate with the players of the computer game Foldit to generate high-quality de novo structural models. This development could greatly speed the generation of excellent cryo-EM structures when used in addition to current methods. Less than a decade ago, before the "resolution revolution," cryo-electron microscopy (cryo-EM) was indulgently called "blobology" . Whereas seminal work of cryo-EM experts resulted in high-resolution 3D maps and atomic models of ordered assemblies such as 2D crystals, helical arrays, and icosahedral viruses , commonly obtained 3D maps of less regular or asymmetric objects could be interpreted only in terms of global 3D architecture, domain organization, and-at most-secondary structure elements. Atomic model building was the privilege and expertise of crystallographers, requiring careful consideration of structural details such as bond geometry, steric clashes, and hydrogen bonds. Now, however, thanks to spectacular progress in both hardware and software, cryo-EM scientists suddenly face the necessity of building atomic models into near-atomic resolution maps. This unanticipated promotion from "blobologists" to "structure solvers" is not as straightforward as it may seem, because model building and refinement are labor-intensive and require expertise in macromolecular