Bridging the resolution gap between x-ray crystallography and electron microscopy

While x-ray crystallography provides atomic resolution structures of proteins and small viruses, electron microscopy can provide complementary structural information on larger assemblies. A significant computational challenge is faced in bridging the resolution gap between the two techniques. X-ray crystallographic data is collected in the range of 2-10 Å, while image reconstructions from electron micrographs are at a resolution of 25-35 Å. A further problem is that density derived from cryo-electron micrographs is distorted by the contrast transfer function of the microscope, whichaccentuates certain resolution bands.A novel combination of electron microscopy and x-ray crystallography has revealed the various structural components forming the capsid of human type 2 adenovirus. An image reconstruction of the intact virus (Fig. 1), derived from cryo-electron micrographs, was deconvolved with an approximate contrast transfer function to mitigate microscope distortions (Fig. 2). A model capsid was calculated from 240 copies of the crystallographic structure of the major capsid protein and filtered to the correct resolution (Fig. 3).

Download Full-text

20 YEARS OF LEPTIN: Insights into signaling assemblies of the leptin receptor

Journal of Endocrinology ◽

10.1530/joe-14-0264 ◽

2014 ◽

Vol 223 (1) ◽

pp. T9-T23 ◽

Cited By ~ 42

Author(s):

Frank Peelman ◽

Lennart Zabeau ◽

Kedar Moharana ◽

Savvas N Savvides ◽

Jan Tavernier

Keyword(s):

Electron Microscopy ◽

Energy Homeostasis ◽

Leptin Receptor ◽

Structural Information ◽

Cell Types ◽

Activation Mechanism ◽

Peripheral Cell ◽

X Ray ◽

X Ray Crystallography ◽

X Ray Scattering

Leptin plays a central role in the control of body weight and energy homeostasis, but is a pleiotropic cytokine with activities on many peripheral cell types. In this review, we discuss the interaction of leptin with its receptor, and focus on the structural and mechanistic aspects of the extracellular aspects of leptin receptor (LR) activation. We provide an extensive overview of all structural information that has been obtained for leptin and its receptor via X-ray crystallography, electron microscopy, small-angle X-ray scattering, homology modeling, and mutagenesis studies. The available knowledge is integrated into putative models toward a recapitulation of the LR activation mechanism.

Download Full-text

The Relation Between the Phase of the Electron Wave (Which is Lost When an Em Image is Recorded) and the Preserved Crystallographic Structure Factor Phases

Microscopy and Microanalysis ◽

10.1017/s1431927600012022 ◽

1997 ◽

Vol 3 (S2) ◽

pp. 1027-1028

Author(s):

Sven Hovmöller ◽

Xiaodong Zou

Keyword(s):

Electron Microscopy ◽

General Solution ◽

Structure Factor ◽

Crystallographic Structure ◽

Phase Problem ◽

Electron Wave ◽

X Ray ◽

Patterson Function ◽

X Ray Crystallography ◽

Mathematical Problems

The phase problem in X-ray crystallography is one of the most interesting and most studied mathematical problems that exist. There is still today no general solution of the phase problem, but partial solutions have resulted in at least 8 Nobel Prizes, even though one of the most prominent solutions, the Patterson function, was not awarded the Prize.It has frequently been claimed that there should be a phase problem also in electron microscopy. It may be more correct to say that there is no phase problem, only a phase confusion problem in electron microscopy. The phase confusion problem has arisen since the word phase is used for (at least) two very different physical entities in the field of electron microscopy. When crystallographers speak about phases, they mean the crystallographic structure factor phases, while physicists mean the wave front phases. In order to resolve the phase confusion problem, it is necessary to define clearly which phases are meant.

Download Full-text

Molecular architecture of the yeast Mediator complex

eLife ◽

10.7554/elife.08719 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 95

Author(s):

Philip J Robinson ◽

Michael J Trnka ◽

Riccardo Pellarin ◽

Charles H Greenberg ◽

David A Bushnell ◽

...

Keyword(s):

Electron Microscopy ◽

Structural Information ◽

Internal Controls ◽

Mediator Complex ◽

Amino Acid Residues ◽

Molecular Architecture ◽

Structural Elements ◽

X Ray ◽

X Ray Crystallography ◽

D Model

The 21-subunit Mediator complex transduces regulatory information from enhancers to promoters, and performs an essential role in the initiation of transcription in all eukaryotes. Structural information on two-thirds of the complex has been limited to coarse subunit mapping onto 2-D images from electron micrographs. We have performed chemical cross-linking and mass spectrometry, and combined the results with information from X-ray crystallography, homology modeling, and cryo-electron microscopy by an integrative modeling approach to determine a 3-D model of the entire Mediator complex. The approach is validated by the use of X-ray crystal structures as internal controls and by consistency with previous results from electron microscopy and yeast two-hybrid screens. The model shows the locations and orientations of all Mediator subunits, as well as subunit interfaces and some secondary structural elements. Segments of 20–40 amino acid residues are placed with an average precision of 20 Å. The model reveals roles of individual subunits in the organization of the complex.

Download Full-text

Electron microscopy of rabbit FC crystals

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077220 ◽

1983 ◽

Vol 41 ◽

pp. 730-731

Author(s):

Robert A. Grant ◽

Laura L. Degn ◽

Wah Chiu ◽

John Robinson

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

High Resolution Electron Microscopy ◽

Molecular Replacement ◽

X Ray ◽

X Ray Crystallography ◽

Resolution Electron ◽

Replacement Technique ◽

Hydrated Crystal ◽

Molecular Structure Analysis

Proteolytic digestion of the immunoglobulin IgG with papain cleaves the molecule into an antigen binding fragment, Fab, and a compliment binding fragment, Fc. Structures of intact immunoglobulin, Fab and Fc from various sources have been solved by X-ray crystallography. Rabbit Fc can be crystallized as thin platelets suitable for high resolution electron microscopy. The structure of rabbit Fc can be expected to be similar to the known structure of human Fc, making it an ideal specimen for comparing the X-ray and electron crystallographic techniques and for the application of the molecular replacement technique to electron crystallography. Thin protein crystals embedded in ice diffract to high resolution. A low resolution image of a frozen, hydrated crystal can be expected to have a better contrast than a glucose embedded crystal due to the larger density difference between protein and ice compared to protein and glucose. For these reasons we are using an ice embedding technique to prepare the rabbit Fc crystals for molecular structure analysis by electron microscopy.

Download Full-text

Ultimate resolution and information in electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086787 ◽

1991 ◽

Vol 49 ◽

pp. 496-497

Author(s):

D. Van Dyck

Keyword(s):

Electron Microscopy ◽

Electron Microscope ◽

Transfer Function ◽

Information Transfer ◽

Communication Channel ◽

Structural Information ◽

Information Rate ◽

Noise Power ◽

Signal Power ◽

Imaging Device

An (electron) microscope can be considered as a communication channel that transfers structural information between an object and an observer. In electron microscopy this information is carried by electrons. According to the theory of Shannon the maximal information rate (or capacity) of a communication channel is given by C = B log2 (1 + S/N) bits/sec., where B is the band width, and S and N the average signal power, respectively noise power at the output. We will now apply to study the information transfer in an electron microscope. For simplicity we will assume the object and the image to be onedimensional (the results can straightforwardly be generalized). An imaging device can be characterized by its transfer function, which describes the magnitude with which a spatial frequency g is transferred through the device, n is the noise. Usually, the resolution of the instrument ᑭ is defined from the cut-off 1/ᑭ beyond which no spadal information is transferred.

Download Full-text

Structure of the Lassa Virus Nucleoprotein Revealed by X-ray Crystallography, Small-angle X-ray Scattering, and Electron Microscopy

Journal of Biological Chemistry ◽

10.1074/jbc.m111.278838 ◽

2011 ◽

Vol 286 (44) ◽

pp. 38748-38756 ◽

Cited By ~ 37

Author(s):

Linda Brunotte ◽

Romy Kerber ◽

Weifeng Shang ◽

Florian Hauer ◽

Meike Hass ◽

...

Keyword(s):

Electron Microscopy ◽

Small Angle ◽

Lassa Virus ◽

X Ray ◽

X Ray Crystallography ◽

X Ray Scattering ◽

Ray Scattering

Download Full-text

Examining the structure of the mature amyloid fibril

Biochemical Society Transactions ◽

10.1042/bst0300521 ◽

2002 ◽

Vol 30 (4) ◽

pp. 521-525 ◽

Cited By ~ 43

Author(s):

O. S. Makin ◽

L. C. Serpell

Keyword(s):

Self Assembly ◽

Rational Design ◽

Amyloid Fibrils ◽

Structural Information ◽

X Ray ◽

X Ray Crystallography ◽

Cryo Electron Microscopy ◽

Fibre Diffraction ◽

Complementary Techniques ◽

Mature Fibril

The pathogenesis of the group of diseases known collectively as the amyloidoses is characterized by the deposition of insoluble amyloid fibrils. These are straight, unbranching structures about 70–120 å (1 å = 0.1 nm) in diameter and of indeterminate length formed by the self-assembly of a diverse group of normally soluble proteins. Knowledge of the structure of these fibrils is necessary for the understanding of their abnormal assembly and deposition, possibly leading to the rational design of therapeutic agents for their prevention or disaggregation. Structural elucidation is impeded by fibril insolubility and inability to crystallize, thus preventing the use of X-ray crystallography and solution NMR. CD, Fourier-transform infrared spectroscopy and light scattering have been used in the study of the mechanism of fibril formation. This review concentrates on the structural information about the final, mature fibril and in particular the complementary techniques of cryo-electron microscopy, solid-state NMR and X-ray fibre diffraction.

Download Full-text