Crystallographic Studies of Biological Macromolecules using Synchrotron Radiation

1988 ◽  
pp. 509-536
Author(s):  
Peter F. Lindley
Keyword(s):  
Synchrotron Radiation ◽  
Biological Macromolecules

scholarly journals Macromolecular crystallography at synchrotron radiation sources: current status and future developments

2010 ◽  
Vol 466 (2124) ◽  
pp. 3421-3452 ◽  
Author(s):  
E. M. H. Duke ◽  
L. N. Johnson
Keyword(s):  
Synchrotron Radiation ◽  
Drug Targets ◽  
Protein Complexes ◽  
Current Status ◽  
Anomalous Scattering ◽  
Biological Macromolecules ◽  
X Rays ◽  
Time Resolved ◽  
X Ray ◽  
Atomic Structures

X-ray diffraction with synchrotron radiation (SR) has revealed the atomic structures of numerous biological macromolecules including proteins and protein complexes, nucleic acids and their protein complexes, viruses, membrane proteins and drug targets. The bright SR X-ray beam with its small divergence has made the study of weakly diffracting crystals of large biological molecules possible. The ability to tune the wavelength of the SR beam to the absorption edge of certain elements has allowed anomalous scattering to be exploited for phase determination. We review the developments at synchrotron sources and beamlines from the early days to the present time, and discuss the significance of the results in providing a deeper understanding of the biological function, the design of new therapeutic molecules and time-resolved studies of dynamic events using pump–probe techniques. Radiation damage, a problem with bright X-ray sources, has been partially alleviated by collecting data at low temperature (100 K) but work is ongoing. In the most recent development, free electron laser sources can offer a peak brightness of hard X-rays approximately 10 8 times brighter than that achieved at SR sources. We describe briefly how early experiments at FLASH and Linear Coherent Light Source have shown exciting possibilities for the future.

scholarly journals Data-collection facility for study of biological macromolecules using synchrotron radiation

1984 ◽  
Vol 40 (a1) ◽  
pp. C10-C10
Author(s):  
R. M. Sweet ◽  
J. B. Hastings ◽  
W. C. Thomlinson ◽  
D. S. Wise ◽  
B. P. Schoenborn
Keyword(s):  
Synchrotron Radiation ◽  
Data Collection ◽  
Biological Macromolecules

scholarly journals Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation

IUCrJ ◽  
2017 ◽  
Vol 4 (4) ◽  
pp. 439-454 ◽  
Author(s):  
Jose M. Martin-Garcia ◽  
Chelsie E. Conrad ◽  
Garrett Nelson ◽  
Natasha Stander ◽  
Nadia A. Zatsepin ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Synchrotron Radiation ◽  
Data Collection ◽  
Structure Determination ◽  
Data Reduction ◽  
High Viscosity ◽  
Proteinase K ◽  
Cubic Phase ◽  
Biological Macromolecules ◽  
Synchrotron Source

Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5–20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A2Aadenosine receptor (A2AAR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suitesCheetahandCrystFELenabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A2AAR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A2AAR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5–20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.

Observation of biological macromolecules using various electron microscopic methods

1978 ◽  
Vol 36 (2) ◽  
pp. 170-171
Author(s):  
Mitsuo Ohtsuki ◽  
Michael Sogard
Keyword(s):  
Electron Microscope ◽  
Phase Contrast ◽  
Image Interpretation ◽  
Density Lipoprotein ◽  
Biological Macromolecules ◽  
Contrast Effects ◽  
Biological Structure ◽  
Electron Microscopic ◽  
Structural Investigations ◽  
Staining Techniques

Structural investigations of biological macromolecules commonly employ CTEM with negative staining techniques. Difficulties in valid image interpretation arise, however, due to problems such as variability in thickness and degree of penetration of the staining agent, noise from the supporting film, and artifacts from defocus phase contrast effects. In order to determine the effects of these variables on biological structure, as seen by the electron microscope, negative stained macromolecules of high density lipoprotein-3 (HDL3) from human serum were analyzed with both CTEM and STEM, and results were then compared with CTEM micrographs of freeze-etched HDL3. In addition, we altered the structure of this molecule by digesting away its phospholipid component with phospholipase A2 and look for consistent changes in structure.

Measurement and Reduction of Radiation Damage in Frozen Hydrated Crystalline Specimens

1978 ◽  
Vol 36 (3) ◽  
pp. 70-77 ◽  
Author(s):  
R.M. Glaeser ◽  
S.B. Hayward
Keyword(s):  
Diffraction Pattern ◽  
Structural Information ◽  
Structural Disorder ◽  
Structural Features ◽  
Biological Macromolecules ◽  
Diffraction Intensity ◽  
Object Structure ◽  
Specimen Material ◽  
Unit Cells ◽  
Identical Unit

Highly ordered or crystalline biological macromolecules become severely damaged and structurally disordered after a brief electron exposure. Evidence that damage and structural disorder are occurring is clearly given by the fading and eventual disappearance of the specimen's electron diffraction pattern. The fading and disappearance of sharp diffraction spots implies a corresponding disappearance of periodic structural features in the specimen. By the same token, there is a oneto- one correspondence between the disappearance of the crystalline diffraction pattern and the disappearance of reproducible structural information that can be observed in the images of identical unit cells of the object structure. The electron exposures that result in a significant decrease in the diffraction intensity will depend somewhat upon the resolution (Bragg spacing) involved, and can vary considerably with the chemical makeup and composition of the specimen material.

Concentration determination of chromatin in unstained resin-embedded sections by means of Z-contrast in STEM

1990 ◽  
Vol 48 (2) ◽  
pp. 186-187
Author(s):  
M. Haider ◽  
B. Bohrmann
Keyword(s):  
Energy Loss ◽  
Freeze Substitution ◽  
Biological Macromolecules ◽  
Local Concentration ◽  
E Coli ◽  
Concentration Determination ◽  
Phosphorous Content ◽  
Z Contrast ◽  
Electron Energy Loss

The technique of Z-contrast in STEM offers the possibility to determine the local concentration of macromolecules like lipids, proteins or DNA. Contrast formation depends on the atomic composition of the particular structure. In the case of DNA, its phosphorous content discriminates it from other biological macromolecules. In our studies, sections of E. coli, the dinoflagellate Amphidinium carterae and Euglena spec. cells were used which were obtained by cryofixation followed by freeze-substitution into acetone with 3% glutaraldehyde. The samples were then embedded either in Lowicryl HM20 at low temperature or in Epon at high temperature. Sections were coated on both sides with 30Å carbon.The DF- and the inelastic image have been recorded simultaneously with a Cryo-STEM. This Cryo-STEM is equipped with a highly dispersive Electron Energy Loss Spectrometer. With this instrument pure Z-contrast can be achieved either with a Filtered DF-image divided by the inelastic image or, as is used in this paper, by dividing the conventional DF-image by an inelastic image which has been recorded with an inelastic detector whose response is dependent on the total energy loss of the inelastically scattered electrons.

Resolution assessment from spectral signal-to-noise ratios

1987 ◽  
Vol 45 ◽  
pp. 746-747
Author(s):  
M. Unser ◽  
B.L. Trus ◽  
A.C. Steven
Keyword(s):  
Electron Microscopy ◽  
Limiting Factor ◽  
Biological Macromolecules ◽  
Signal To Noise ◽  
Differential Phase ◽  
Traditional Measure ◽  
Spectral Signal ◽  
Two Measures ◽  
Diffraction Patterns ◽  
Averaging Techniques

Since the resolution-limiting factor in electron microscopy of biological macromolecules is not instrumental, but is rather the preservation of structure, operational definitions of resolution have to be based on the mutual consistency of a set of like images. The traditional measure of resolution for crystalline specimens in terms of the extent of periodic reflections in their diffraction patterns is such a criterion. With the advent of correlation averaging techniques for lattice rectification and the analysis of non-crystalline specimens, a more general - and desirably, closely compatible - resolution criterion is needed. Two measures of resolution for correlation-averaged images have been described, namely the differential phase residual (DPR) and the Fourier ring correlation (FRC). However, the values that they give for resolution often differ substantially. Furthermore, neither method relates in a straightforward way to the long-standing resolution criterion for crystalline specimens.

A TEM study of electron tunneling in biological macromolecules

1987 ◽  
Vol 45 ◽  
pp. 140-141
Author(s):  
J. A. Panitz
Keyword(s):  
Stark Effect ◽  
Electron Tunneling ◽  
Imaging Modality ◽  
Tunneling Current ◽  
Biological Species ◽  
Scanning Tunneling ◽  
Biological Macromolecules ◽  
Flat Surfaces ◽  
Virus Particles ◽  
Stm Images

Tunneling is a ubiquitous phenomenon. Alpha particle disintegration, the Stark effect, superconductivity in thin films, field-emission, and field-ionization are examples of electron tunneling phenomena. In the scanning tunneling microscope (STM) electron tunneling is used as an imaging modality. STM images of flat surfaces show structure at the atomic level. However, STM images of large biological species deposited onto flat surfaces are disappointing. For example, unstained virus particles imaged in the STM do not resemble their TEM counterparts.It is not clear how an STM image of a biological species is formed. Most biological species are large compared to the nominal electrode separation of ∼ 1nm that is required for electron tunneling. To form an image of a biological species, the tunneling electrodes must be separated by a distance that would normally be too large for a tunneling current to be observed.

Depletion and Reconstitution of Active E. Coli Ribosomes

1975 ◽  
Vol 33 ◽  
pp. 640-641
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Protein Biosynthesis ◽  
Large Subunit ◽  
High Resolution Electron Microscopy ◽  
Small Subunit ◽  
Structure And Function ◽  
Biologically Active ◽  
Biological Macromolecules ◽  
E Coli ◽  
Resolution Electron ◽  
And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

Sign in / Sign up

Export Citation Format

Share Document