Cryo-EM of macromolecular assemblies at near-atomic resolution

Cryo-EM has revealed the structures of many challenging yet exciting macromolecular assemblies at near-atomic resolution (3–4.5Å), providing biological phenomena with molecular descriptions. However, at these resolutions, accurately positioning individual atoms remains challenging and error-prone. Manually refining thousands of amino acids – typical in a macromolecular assembly – is tedious and time-consuming. We present an automated method that can improve the atomic details in models that are manually built in near-atomic-resolution cryo-EM maps. Applying the method to three systems recently solved by cryo-EM, we are able to improve model geometry while maintaining the fit-to-density. Backbone placement errors are automatically detected and corrected, and the refinement shows a large radius of convergence. The results demonstrate that the method is amenable to structures with symmetry, of very large size, and containing RNA as well as covalently bound ligands. The method should streamline the cryo-EM structure determination process, providing accurate and unbiased atomic structure interpretation of such maps.

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Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta

10.1101/050286 ◽

2016 ◽

Author(s):

Ray Yu-Ruei Wang ◽

Yifan Song ◽

Benjamin A Barad ◽

Yifan Cheng ◽

James S Fraser ◽

...

Keyword(s):

Structure Refinement ◽

Atomic Resolution ◽

Large Radius ◽

Macromolecular Assemblies ◽

Automated Method ◽

Large Size ◽

Biological Phenomena ◽

Determination Process ◽

Improve Model ◽

Model Geometry

AbstractCryo-EM has revealed many challenging yet exciting macromolecular assemblies at near-atomic resolution (3-4.5Å), providing biological phenomena with molecular descriptions. However, at these resolutions accurately positioning individual atoms remains challenging and may be error-prone. Manually refining thousands of amino acids – typical in a macromolecular assembly – is tedious and time-consuming. We present an automated method that can improve the atomic details in models manually built in near-atomic-resolution cryo-EM maps. Applying the method to three systems recently solved by cryo-EM, we are able to improve model geometry while maintaining or improving the fit-to-density. Backbone placement errors are automatically detected and corrected, and the refinement shows a large radius of convergence. The results demonstrate the method is amenable to structures with symmetry, of very large size, and containing RNA as well as covalently bound ligands. The method should streamline the cryo-EM structure determination process, providing accurate and unbiased atomic structure interpretation of such maps.

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Integrative Modeling of Macromolecular Assemblies from Low to Near-Atomic Resolution

Computational and Structural Biotechnology Journal ◽

10.1016/j.csbj.2015.08.005 ◽

2015 ◽

Vol 13 ◽

pp. 492-503 ◽

Cited By ~ 18

Author(s):

Xiaojun Xu ◽

Chunli Yan ◽

Robert Wohlhueter ◽

Ivaylo Ivanov

Keyword(s):

Atomic Resolution ◽

Macromolecular Assemblies ◽

Integrative Modeling

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Introduction to Cell Membranes at Molecular Resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100070217 ◽

1978 ◽

Vol 36 (3) ◽

pp. 497-502

Author(s):

R.J. Barrnett

Keyword(s):

Cell Membranes ◽

International Federation ◽

Mountain Range ◽

Cellular Membranes ◽

Short History ◽

Biological Organization ◽

Macromolecular Assemblies ◽

The Past

This subject, is like observing the panorama of a mountain range, magnificent towering peaks, but it doesn't take much duration of observation to recognize that they are still in the process of formation. The mountains consist of approaches, materials and methods and the rocky substance of information has accumulated to such a degree that I find myself concentrating on the foothills in the foreground in order to keep up with the advance; the edifices behind form a wonderous, substantive background. It's a short history for such an accumulation and much of it has been moved by the members of the societies that make up this International Federation. My panel of speakers are here to provide what we hope is an interesting scientific fare, based on the fact that there is a continuum of biological organization from biochemical molecules through macromolecular assemblies and cellular membranes to the cell itself. Indeed, this fact explains the whole range of towering peaks that have emerged progressively during the past 25 years.

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Effect of strain on ADF-STEM high-resolution images

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100087641 ◽

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010008701x ◽

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.

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Scanning tunneling microscopy of E. coli RNA polymerase deposited onto an Au substrate by an electrochemical process

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086192 ◽

1991 ◽

Vol 49 ◽

pp. 378-379

Author(s):

Rebecca W. Keller ◽

Carlos Bustamante ◽

David Bear

Keyword(s):

Scanning Tunneling Microscope ◽

Biological Sample ◽

Substrate Surface ◽

Electrochemical Process ◽

Atomic Resolution ◽

Scanning Tunneling ◽

Tunneling Microscopy ◽

Tunneling Microscope ◽

E Coli ◽

Preparation Techniques

Under ideal conditions, the Scanning Tunneling Microscope (STM) can create atomic resolution images of different kinds of samples. The STM can also be operated in a variety of non-vacuum environments. Because of its potentially high resolution and flexibility of operation, it is now being applied to image biological systems. Several groups have communicated the imaging of double and single stranded DNA.However, reproducibility is still the main problem with most STM results on biological samples. One source of irreproducibility is unreliable sample preparation techniques. Traditional deposition methods used in electron microscopy, such as glow discharge and spreading techniques, do not appear to work with STM. It seems that these techniques do not fix the biological sample strongly enough to the substrate surface. There is now evidence that there are strong forces between the STM tip and the sample and, unless the sample is strongly bound to the surface, it can be swept aside by the tip.

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Multimode light microscopy and fluorescence-based reagents as tools for the study of chemical and molecular dynamics of living cells and tissues

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122496 ◽

1992 ◽

Vol 50 (1) ◽

pp. 418-419

Author(s):

D. L. Taylor

Keyword(s):

Living Cells ◽

Cellular Level ◽

Molecular Techniques ◽

Cellular Functions ◽

Macromolecular Assemblies ◽

Cell Functions ◽

Molecular Events ◽

Yield Information ◽

Full Knowledge ◽

Structural Methods

Cells function through the complex temporal and spatial interplay of ions, metabolites, macromolecules and macromolecular assemblies. Biochemical approaches allow the investigator to define the components and the solution chemical reactions that might be involved in cellular functions. Static structural methods can yield information concerning the 2- and 3-D organization of known and unknown cellular constituents. Genetic and molecular techniques are powerful approaches that can alter specific functions through the manipulation of gene products and thus identify necessary components and sequences of molecular events. However, full knowledge of the mechanism of particular cell functions will require direct measurement of the interplay of cellular constituents. Therefore, there has been a need to develop methods that can yield chemical and molecular information in time and space in living cells, while allowing the integration of information from biochemical, molecular and genetic approaches at the cellular level.

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