Solving the structure of macromolecular complexes using transmission electron microscopy can be an arduous task. Many of the steps in this process rely strongly on the aid of pre-existing structural ...knowledge, and are greatly complicated when this information is unavailable. Here, we present two software tools meant to facilitate particle picking, an early stage in the single-particle processing of unknown macromolecules. The first tool, DoG Picker, is an efficient and reasonably general, particle picker based on the Difference of Gaussians (DoG) image transform. It can function alone, as a reference-free particle picker with the unique ability to sort particles based on size, or it can also be used as a way to bootstrap the creation of templates or training datasets for other particle pickers. The second tool is TiltPicker, an interactive graphical interface application designed to streamline the selection of particle pairs from tilted-pair datasets. In many respects, TiltPicker is a re-implementation of the SPIDER WEB tilted-particle picker, but built on modern computer frameworks making it easier to deploy and maintain. The TiltPicker program also includes several useful new features beyond those of its predecessor.
Summary
Although high‐resolution single‐particle cryo‐electron microscopy (cryo‐EM) is now producing a rapid stream of breakthroughs in structural biology, it nevertheless remains the case that the ...preparation of suitable frozen‐hydrated samples on electron microscopy grids is often quite challenging. Purified samples that are intact and structurally homogeneous – while still in the test tube – may not necessarily survive the standard methods of making extremely thin, aqueous films on grids. As a result, it is often necessary to try a variety of experimental conditions before finally finding an approach that is optimal for the specimen at hand. Here, we summarize some of our collective experiences to date in optimizing sample preparation, in the hope that doing so will be useful to others, especially those new to the field. We also hope that an open discussion of these common challenges will encourage the development of more generally applicable methodology. Our collective experiences span a diverse range of biochemical samples and most of the commonly used variations in how grids are currently prepared. Unfortunately, none of the currently used optimization methods can be said, in advance, to be the one that ultimately will work when a project first begins. Nevertheless, there are some preferred first steps to explore when facing specific problems that can be more generally recommended, based on our experience and that of many others in the cryo‐EM field.
The new generation of direct electron detectors has been a major contributor to the recent resolution revolution in cryo-electron microscopy. Optimal use of these new cameras using automated data ...collection software is critical for high-throughput near-atomic resolution cryo-electron microscopy research. We present an overview of the practical aspects of automated data collection in the context of this new generation of direct detectors, highlighting the differences, challenges, and opportunities the new detectors provide compared to the previous generation of data acquisition media.
Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been ...extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region (the neck linker) in kinesin using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy. This region becomes immobilized and extended towards the microtubule 'plus' end when kinesin binds microtubules and ATP, and reverts to a more mobile conformation when gamma-phosphate is released after nucleotide hydrolysis. This conformational change explains both the direction of kinesin motion and processive movement by the kinesin dimer.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Myosins and kinesins are molecular motors that hydrolyse ATP to track along
actin filaments and microtubules, respectively. Although the kinesin family
includes motors that move towards either the ...plus or minus ends of microtubules, all characterized myosin motors move towards the barbed (+) end
of actin filaments. Crystal structures of myosin II (refs 3,4,5,6) have shown that small movements within the myosin motor
core are transmitted through the 'converter domain' to a 'lever
arm' consisting of a light-chain-binding helix and associated light
chains. The lever arm further amplifies the motions of the
converter domain into large directed movements. Here
we report that myosin VI, an unconventional myosin,
moves towards the pointed (-) end of actin. We visualized the myosin VI construct
bound to actin using cryo-electron microscopy and image analysis, and found
that an ADP-mediated conformational change in the domain distal to the motor,
a structure likely to be the effective lever arm, is in the opposite direction
to that observed for other myosins. Thus, it appears that myosin VI achieves
reverse-direction movement by rotating its lever arm in the opposite direction
to conventional myosin lever arm movement.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
We report on a genomic and functional analysis of a novel marine siphovirus, the Vibrio phage SIO‐2. This phage is lytic for related Vibrio species of great ecological interest including the ...broadly antagonistic bacterium Vibrio sp. SWAT3 as well as notable members of the Harveyi clade (V. harveyi ATTC BAA‐1116 and V. campbellii ATCC 25920). Vibrio phage SIO‐2 has a circularly permuted genome of 80 598 bp, which displays unusual features. This genome is larger than that of most known siphoviruses and only 38 of the 116 predicted proteins had homologues in databases. Another divergence is manifest by the origin of core genes, most of which share robust similarities with unrelated viruses and bacteria spanning a wide range of phyla. These core genes are arranged in the same order as in most bacteriophages but they are unusually interspaced at two places with insertions of DNA comprising a high density of uncharacterized genes. The acquisition of these DNA inserts is associated with morphological variation of SIO‐2 capsid, which assembles as a large (80 nm) shell with a novel T = 12 symmetry. These atypical structural features confer on SIO‐2 a remarkable stability to a variety of physical, chemical and environmental factors. Given this high level of functional and genomic novelty, SIO‐2 emerges as a model of considerable interest in ecological and evolutionary studies.
Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when a very large number of images are required to achieve ...three-dimensional reconstructions at near atomic resolution. Investigation of fast, accurate approaches for automatic particle detection has become one of the current challenges in the cryoEM community. At the same time, the investigation is hampered by the fact that few benchmark particles or image datasets exist in the community. The unavailability of such data makes it difficult to evaluate newly developed algorithms and to leverage expertise from other disciplines. The paper presents our recent contribution to this effort. It also describes our newly developed computational framework for particle detection, through the application of edge detection and a sequence of ordered Hough transforms. Experimental results using keyhole limpet hemocyanin (KLH) as a model particle are very promising. In addition, it introduces a newly established web site, designed to support the investigation of automatic particle detection by providing an annotated image dataset of KLH available to the general scientific community.
We have used electron tomography to characterize the self-assembly of nanostructures formed by rodcoil copolymers. The rodcoil copolymers used contained a perfectly monodisperse rod segment prepared ...by stepwise synthesis. The chemical compound rod segment is, in turn, covalently linked at one terminus to an end-functionalized polyisoprene segment prepared by living polymerization. We found that rodcoil molecules with rod volume fraction equal to 0.36 self-assemble into long strips with a nanoscale cross section. At lower rod volume fractions, the rodcoil molecules self-assemble into supramolecular aggregates with nanoscale x,y,z dimensions. Interestingly, the nanostructures organize into discrete layers of uniform thickness, containing in some cases a hexagonal 2D superlattice of rodcoil aggregates. Furthermore, nanostructures were found to self-assemble in all cases with three-dimensional order across the layers. The unique three-dimensional order observed in multilayers of the nanostructures must originate in the anisotropic aggregation of rod segments and the consequent space filling requirements as well as coil entropic penalties.
A three-dimensional analysis of the nuclear pore complex reveals the underlying, highly symmetric framework of this supramolecular assembly, how it is anchored in the nuclear membrane, and how it is ...built from many distinct, interconnected subunits. The arrangement of the subunits within the membrane pore creates a large central channel, through which active nucleocytoplasmic transport is known to occur, and eight smaller peripheral channels that are probable routes for passive diffusion of ions and small molecules.
We report here on the current state of our efforts in automated molecular microscopy. Our primary automated data acquisition software system, Leginon, has been completely redesigned over the past two ...years. The new distributed system has been developed using the Python programming language and is compatible with both Linux and Windows operating systems. The new flexible architecture was designed to allow for the development of customized data collection protocols, several of which are described here. The system has been used to acquire data for ∼150 experiments and we have demonstrated the capacity for high throughput data acquisition by acquiring images of more than 100
000 particles in a single session at the microscope.
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