scholarly journals Separating the effects of nucleotide and EB binding on microtubule structure

2018 ◽  
Vol 115 (27) ◽  
pp. E6191-E6200 ◽  
Author(s):  
Rui Zhang ◽  
Benjamin LaFrance ◽  
Eva Nogales
Keyword(s):  
High Resolution ◽  
Binding Proteins ◽  
Dynamic Instability ◽  
Stable State ◽  
Structural Data ◽  
Atomic Resolution ◽  
Mechanical Forces ◽  
Gtp Hydrolysis ◽  
Associated Proteins ◽  
Microtubule Structure

Microtubules (MTs) are polymers assembled from αβ-tubulin heterodimers that display the hallmark behavior of dynamic instability. MT dynamics are driven by GTP hydrolysis within the MT lattice, and are highly regulated by a number of MT-associated proteins (MAPs). How MAPs affect MTs is still not fully understood, partly due to a lack of high-resolution structural data on undecorated MTs, which need to serve as a baseline for further comparisons. Here we report three structures of MTs in different nucleotide states (GMPCPP, GDP, and GTPγS) at near-atomic resolution and in the absence of any binding proteins. These structures allowed us to differentiate the effects of nucleotide state versus MAP binding on MT structure. Kinesin binding has a small effect on the extended, GMPCPP-bound lattice, but hardly affects the compacted GDP-MT lattice, while binding of end-binding (EB) proteins can induce lattice compaction (together with lattice twist) in MTs that were initially in an extended and more stable state. We propose a MT lattice-centric model in which the MT lattice serves as a platform that integrates internal tubulin signals, such as nucleotide state, with outside signals, such as binding of MAPs or mechanical forces, resulting in global lattice rearrangements that in turn affect the affinity of other MT partners and result in the exquisite regulation of MT dynamics.

scholarly journals Structural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubules

2021 ◽  
Author(s):  
Benjamin J LaFrance ◽  
Johanna Roostalu ◽  
Gil Henkin ◽  
Basil J Greber ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Amino Acids ◽  
High Resolution ◽  
Conformational Changes ◽  
Binding Proteins ◽  
Dynamic Instability ◽  
Structural Transitions ◽  
Gtp Hydrolysis ◽  
Tirf Microscopy ◽  
Mechanistic Insight ◽  
Catalytically Active

Microtubules (MTs) are polymers of alphabeta-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes, and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here we use cryo-EM and TIRF microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice, but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wildtype GDP-MTs. End binding proteins of the EB family have the ability to compact both mutant GTP-lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wildtype MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these first high-resolution structures of truly GTP-bound MTs add mechanistic insight to our understanding of MT dynamic instability.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

scholarly journals Abstract P-33: Structural Basis for the Growing Microtubule End Recognition by End Binding Proteins

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S26-S26
Author(s):  
Alena Korshunova
Keyword(s):  
Amino Acids ◽  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Molecular Mechanism ◽  
Binding Proteins ◽  
Dynamic Instability ◽  
3D Structure ◽  
Structural Basis ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis

Background: Eukaryotic end binding proteins (EBs) can follow the growing microtubule end. EBs play a crucial role in microtubule dynamic instability and promote simultaneously growth rate and catastrophe frequency. It makes EB-like proteins perspective drag targets for a wide number of diseases. But the molecular mechanism of tip tracking by EB-like proteins remains unknown. Studies of mutants have revealed that the conservative amino acid Q102 (numbering relative to the human EB1 protein) plays a key role in the recognition of the growing microtubule end. However, the 3D structure studies revealed that this amino acid has no bonds with tubulin. In this work, we performed structural and phylogenetic analysis of EBs proteins to identify a possible molecular mechanism behind the plus end tracking. Methods: UCSF Chimera10 was used for structural analysis. Phylogenetic analysis was performed with MEGA X software. 3D structures of EBs and microtubules with different states of GTP hydrolysis were used (pdb 3JAK, 3JAS, 3JAT, 3JAW, 3JAL, 3JAR, 6DPU, 6DPV, 6DPW). Results: We have shown that two conservative amino acids (K100, E106) should play an important role in the recognition of the microtubule plus end in addition to Q102. It was concluded that these amino acids together form the plus-end «navigation site» of EBs. Analysis of possible interaction of the «navigation site» amino acids with microtubules in different conformational states suggested that the main mechanism of growing microtubule end recognition is not due to an affinity increase for a certain state of tubulin in microtubules at their end, but it due to a significant affinity decrease in other parts of the microtubule as a result of steric clashes. Conclusion: Thus, the results of the analysis suggested the possible molecular mechanism that provides the tip tracking by EB-like proteins and allowed us to identify the key amino acids of this mechanism.

scholarly journals Structural insights into filament recognition by cellular actin markers

2019 ◽  
Author(s):  
Archana Kumari ◽  
Shubham Kesarwani ◽  
Manjunath G Javoor ◽  
Kutti R. Vinothkumar ◽  
Minhajuddin Sirajuddin
Keyword(s):  
High Resolution ◽  
Binding Sites ◽  
Binding Proteins ◽  
Structural Model ◽  
Structural Data ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Electron Cryomicroscopy ◽  
Structural Insights ◽  
Structural Perspective

AbstractCellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here we describe the electron cryomicroscopy structures of phalloidin, lifeAct and utrophin bound to F-actin, providing the first high-resolution structures and comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce conformations and lifeAct specifically recognizes ADP-actin state, which can be used as a sensor for distinguishing different nucleotide states of F-actin. The utrophin structural model aided designing minimal utrophin, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin binding proteins. Further offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.

Effect of strain on ADF-STEM high-resolution images

1991 ◽  
Vol 49 ◽  
pp. 666-667
Author(s):  
M. Kelly ◽  
D.M. Bird
Keyword(s):  
High Resolution ◽  
Strong Influence ◽  
Intensity Variation ◽  
Dark Field ◽  
Atomic Resolution ◽  
Strain Fields ◽  
High Resolution Image ◽  
Annular Dark Field ◽  
High Resolution Images ◽  
Interesting Alternative

It is well known that strain fields can have a strong influence on the details of HREM images. This, for example, can cause problems in the analysis of edge-on interfaces between lattice mismatched materials. An interesting alternative to conventional HREM imaging has recently been advanced by Pennycook and co-workers where the intensity variation in the annular dark field (ADF) detector is monitored as a STEM probe is scanned across the specimen. It is believed that the observed atomic-resolution contrast is correlated with the intensity of the STEM probe at the atomic sites and the way in which this varies as the probe moves from cell to cell. As well as providing a directly interpretable high-resolution image, there are reasons for believing that ADF-STEM images may be less suseptible to strain than conventional HREM. This is because HREM images arise from the interference of several diffracted beams, each of which is governed by all the excited Bloch waves in the crystal.

Electron crystallographic determination of the 3-D structure of staurolite at atomic resolution

1991 ◽  
Vol 49 ◽  
pp. 540-541
Author(s):  
Kenneth H. Downing ◽  
Hu Meisheng ◽  
Hans-Rudolf Went ◽  
Michael A. O'Keefe
Keyword(s):  
High Resolution ◽  
Three Dimensional ◽  
High Resolution Electron Microscopy ◽  
Atomic Resolution ◽  
Dimensional Structure ◽  
Structure Information ◽  
Resolution Electron ◽  
Scattering Effects ◽  
High Resolution Images ◽  
Effects Of Radiation

With current advances in electron microscope design, high resolution electron microscopy has become routine, and point resolutions of better than 2Å have been obtained in images of many inorganic crystals. Although this resolution is sufficient to resolve interatomic spacings, interpretation generally requires comparison of experimental images with calculations. Since the images are two-dimensional representations of projections of the full three-dimensional structure, information is invariably lost in the overlapping images of atoms at various heights. The technique of electron crystallography, in which information from several views of a crystal is combined, has been developed to obtain three-dimensional information on proteins. The resolution in images of proteins is severely limited by effects of radiation damage. In principle, atomic-resolution, 3D reconstructions should be obtainable from specimens that are resistant to damage. The most serious problem would appear to be in obtaining high-resolution images from areas that are thin enough that dynamical scattering effects can be ignored.

High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

1992 ◽  
Vol 50 (1) ◽  
pp. 512-513
Author(s):  
J. Jakana ◽  
M.F. Schmid ◽  
P. Matsudaira ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Binding Proteins ◽  
Actin Binding ◽  
Eukaryotic Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Actin Bundle ◽  
Actin Bundles ◽  
3 Dimensional ◽  
Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

Tubulin structure at intermediate resolution

1995 ◽  
Vol 53 ◽  
pp. 852-853
Author(s):  
K. H. Downing ◽  
S. G. Wolf ◽  
E. Nogales
Keyword(s):  
High Resolution ◽  
Structural Data ◽  
Therapeutic Drug ◽  
Zinc Ions ◽  
Two Dimensional ◽  
X Ray Diffraction ◽  
X Ray ◽  
Negative Stain ◽  
Functional Mechanisms ◽  
Tubulin Structure

Microtubules are involved in a host of critical cell activities, many of which involve transport of organelles through the cell. Different sets of microtubules appear to form during the cell cycle for different functions. Knowledge of the structure of tubulin will be necessary in order to understand the various functional mechanisms of microtubule assemble, disassembly, and interaction with other molecules, but tubulin has so far resisted crystallization for x-ray diffraction studies. Fortuitously, in the presence of zinc ions, tubulin also forms two-dimensional, crystalline sheets that are ideally suited for study by electron microscopy. We have refined procedures for forming the sheets and preparing them for EM, and have been able to obtain high-resolution structural data that sheds light on the formation and stabilization of microtubules, and even the interaction with a therapeutic drug.Tubulin sheets had been extensively studied in negative stain, demonstrating that the same protofilament structure was formed in the sheets and microtubules. For high resolution studies, we have found that the sheets embedded in either glucose or tannin diffract to around 3 Å.

Atomic resolution characterisation of interface structures by Electron Energy Loss Spectroscopy

1993 ◽  
Vol 51 ◽  
pp. 576-577
Author(s):  
N. D. Browning ◽  
M. M. McGibbon ◽  
M. F. Chisholm ◽  
S. J. Pennycook
Keyword(s):  
High Resolution ◽  
Energy Loss ◽  
Electron Energy ◽  
Electron Energy Loss Spectroscopy ◽  
Imaging Technique ◽  
Atomic Scale ◽  
Atomic Resolution ◽  
Reference Image ◽  
The Impact ◽  
Electron Energy Loss

The recent development of the Z-contrast imaging technique for the VG HB501 UX dedicated STEM, has added a high-resolution imaging facility to a microscope used mainly for microanalysis. This imaging technique not only provides a high-resolution reference image, but as it can be performed simultaneously with electron energy loss spectroscopy (EELS), can be used to position the electron probe at the atomic scale. The spatial resolution of both the image and the energy loss spectrum can be identical, and in principle limited only by the 2.2 Å probe size of the microscope. There now exists, therefore, the possibility to perform chemical analysis of materials on the scale of single atomic columns or planes.In order to achieve atomic resolution energy loss spectroscopy, the range over which a fast electron can cause a particular excitation event, must be less than the interatomic spacing. This range is described classically by the impact parameter, b, which ranges from ~10 Å for the low loss region of the spectrum to <1Å for the core losses.

HRTEM study of valleriite, a hydroxide-bearing copper sulfide

1990 ◽  
Vol 48 (4) ◽  
pp. 462-463
Author(s):  
S. Wang ◽  
P. R. Buseck
Keyword(s):  
Electron Microscope ◽  
High Resolution ◽  
Copper Sulfide ◽  
Carbonaceous Chondrite ◽  
Structural Data ◽  
Basic Structure ◽  
Long Period ◽  
Selected Area Electron Diffraction ◽  
Transmission Electron ◽  
Wavy Structure

Valleriite is an unusual mineral, consisting of intergrowths of sulfide layers (corresponding in structure to the mineral smythite - Fe9S11) and hydroxide layers (corresponding to brucite - Mg(OH2)). It has a composition of approximately 1.526[Mg.68Al.32(OH)2].[Fe1.07Cu.93S2] and consists of two interpenetrating lattices, each of which retains its individual structural and diffraction characteristics parallel to the layering. The valleriite structure is related to that of tochilinite, an unusual iron-rich mineral that is of considerable interest for the origin of certain carbonaceous chondrite meteorites and to those of franckeite and cylindrite, two minerals that are of interest because of their unique morphological and crystallographic properties, e.g., the distinctive curved form of cylindrite and the perfect mica-like cleavage with unusual striations and the long-period wavy structure of franckeite.Our selected-area electron diffraction (SAED) patterns and high-resolution transmission electron microscope (HRTEM) images of valleriite provide new structural data. A basic structure and a new superstructure have been observed.

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