scholarly journals Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

IUCrJ ◽  
2020 ◽  
Vol 7 (2) ◽  
pp. 306-323 ◽  
Author(s):  
Alexander M. Wolff ◽  
Iris D. Young ◽  
Raymond G. Sierra ◽  
Aaron S. Brewster ◽  
Michael W. Martynowycz ◽  
...  
Keyword(s):  
Electron Diffraction ◽  
Structural Information ◽  
Biological Macromolecules ◽  
Structural Studies ◽  
Macromolecular Crystallography ◽  
Delivery Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Physical Conditions ◽  
Serial Crystallography

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

scholarly journals Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

2019 ◽  
Author(s):  
Alexander M Wolff ◽  
Iris D Young ◽  
Raymond G Sierra ◽  
Aaron S Brewster ◽  
Michael W Martynowycz ◽  
...  
Keyword(s):  
Electron Diffraction ◽  
Structural Information ◽  
Biological Macromolecules ◽  
Structural Studies ◽  
Macromolecular Crystallography ◽  
Delivery Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Physical Conditions ◽  
Serial Crystallography

AbstractInnovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometer to micron scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges not encountered in traditional macromolecular crystallography experiments. Here, we describe XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A (CypA). Our results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample preparation and delivery methods required for each type of experiment effect the crystal structure of the enzyme.

scholarly journals New light on protein structure: Macromolecular crystallography

2007 ◽  
Vol 29 (4) ◽  
pp. 32-35
Author(s):  
Armin Wagner
Keyword(s):  
Light Source ◽  
Structural Information ◽  
Considerable Change ◽  
Imperial College ◽  
Atomic Resolution ◽  
Biological Macromolecules ◽  
Macromolecular Crystallography ◽  
X Ray Diffraction ◽  
X Ray ◽  
Structure Solution

X-ray diffraction is the method of choice to determine structural information from biological mac romolecules to atomic resolution. This technique depends on the availability of single crystals of protein, which are notoriously difficult to produce. It can take months or even years to find crystal lization conditions capable of producing crystals with sufficient diffraction quality. During the last few years the field of MX (macromolecular crystallography) has undergone considerable change and most of the steps from protein expression to structure solution have been automated, speeding up the process significantly. Facilities such as Diamond Light Source, the new UK synchrotron radia tion source in Oxfordshire, have been developed to incorporate new automation technologies and Diamond will provide an important user resource for XRD (X-ray diffraction) experiments on crystals of biological macromolecules. Furthermore, in collaboration with Professor So Iwata (Imperial College and Diamond Light Source) and funded by the Wellcome Trust, Diamond Light Source is developing a laboratory dedicated specifically to solving the structure of membrane proteins, the crystallization of which poses a particular problem to the crystallographer.

scholarly journals Illuminating the Secrets of Crystals - Microcrystal Electron Diffraction in Structural Biology

2018 ◽  
Author(s):  
Rob Barringer ◽  
Thomas Meier
Keyword(s):  
Electron Diffraction ◽  
Structural Biology ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Novel Method ◽  
Practical Level ◽  
Crystallographic Theory

An exploration of the crystallographic theory of the relatively novel method of Microcrystal Electron Diffraction (MicroED), via comparison to X-ray crystallography at the theoretical and practical level as it applies to biological macromolecules. We then attempt to outline the limitations and challenges that the technique currently faces in structural biology, and suggest future areas of study that may improve and optimize the technique.

scholarly journals Four-dimensional NOE-NOE spectroscopy of SARS-CoV-2 Main Protease to facilitate resonance assignment and structural analysis

2021 ◽  
Author(s):  
Angus J. Robertson ◽  
Jinfa Ying ◽  
Ad Bax
Keyword(s):  
Resonance Assignment ◽  
Structural Information ◽  
Structural Features ◽  
Structural Studies ◽  
Dipolar Relaxation ◽  
X Ray ◽  
X Ray Crystallography ◽  
Main Protease ◽  
4D Noesy ◽  
Exchange Broadening

Abstract. Resonance assignment and structural studies of larger proteins by NMR can be challenging when exchange broadening, multiple stable conformations, and back-exchanging the fully deuterated chain pose problems. These difficulties arise for the SARS-CoV-2 Main Protease, a homodimer of 2×306 residues. We demonstrate that the combination of four-dimensional (4D) TROSY-NOESY-TROSY spectroscopy and 4D NOESY-NOESY-TROSY spectroscopy provides an effective tool for delineating the 1H-1H dipolar relaxation network. In combination with detailed structural information obtained from prior X-ray crystallography work, such data are particularly useful for extending and validating resonances assignments, as well as for probing structural features.

scholarly journals Where is crystallography going?

2018 ◽  
Vol 74 (2) ◽  
pp. 152-166 ◽  
Author(s):  
Jonathan M. Grimes ◽  
David R. Hall ◽  
Alun W. Ashton ◽  
Gwyndaf Evans ◽  
Robin L. Owen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Synchrotron Radiation ◽  
Electron Diffraction ◽  
Recombinant Proteins ◽  
Free Electron Lasers ◽  
Macromolecular Crystallography ◽  
Signal To Noise ◽  
X Ray ◽  
X Ray Crystallography ◽  
Order Of Magnitude

Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X-ray free-electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X-ray crystallography in rude health, or will imaging methods, especially single-particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide.

scholarly journals Sample Delivery Media for Serial Crystallography

2019 ◽  
Vol 20 (5) ◽  
pp. 1094 ◽  
Author(s):  
Ki Nam
Keyword(s):  
Radiation Damage ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Interaction Point ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
Serial Crystallography ◽  
Serial Femtosecond Crystallography ◽  
Delivery Medium

X-ray crystallographic methods can be used to visualize macromolecules at high resolution. This provides an understanding of molecular mechanisms and an insight into drug development and rational engineering of enzymes used in the industry. Although conventional synchrotron-based X-ray crystallography remains a powerful tool for understanding molecular function, it has experimental limitations, including radiation damage, cryogenic temperature, and static structural information. Serial femtosecond crystallography (SFX) using X-ray free electron laser (XFEL) and serial millisecond crystallography (SMX) using synchrotron X-ray have recently gained attention as research methods for visualizing macromolecules at room temperature without causing or reducing radiation damage, respectively. These techniques provide more biologically relevant structures than traditional X-ray crystallography at cryogenic temperatures using a single crystal. Serial femtosecond crystallography techniques visualize the dynamics of macromolecules through time-resolved experiments. In serial crystallography (SX), one of the most important aspects is the delivery of crystal samples efficiently, reliably, and continuously to an X-ray interaction point. A viscous delivery medium, such as a carrier matrix, dramatically reduces sample consumption, contributing to the success of SX experiments. This review discusses the preparation and criteria for the selection and development of a sample delivery medium and its application for SX.

scholarly journals Illuminating the Secrets of Crystals - Microcrystal Electron Diffraction in Structural Biology

2018 ◽  
Author(s):  
Rob Barringer ◽  
Thomas Meier
Keyword(s):  
Electron Diffraction ◽  
Structural Biology ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Novel Method ◽  
Practical Level ◽  
Crystallographic Theory

An exploration of the crystallographic theory of the relatively novel method of Microcrystal Electron Diffraction (MicroED), via comparison to X-ray crystallography at the theoretical and practical level as it applies to biological macromolecules. We then attempt to outline the limitations and challenges that the technique currently faces in structural biology, and suggest future areas of study that may improve and optimize the technique.

Using image analysis for automated crystal positioning in a synchrotron X-ray beam for high-throughput macromolecular crystallography

2004 ◽  
Vol 37 (2) ◽  
pp. 265-269 ◽  
Author(s):  
Philippe Andrey ◽  
Bernard Lavault ◽  
Florent Cipriani ◽  
Yves Maurin
Keyword(s):  
Image Analysis ◽  
High Throughput ◽  
Protein Crystals ◽  
Structural Studies ◽  
Macromolecular Crystallography ◽  
Absolute Size ◽  
X Ray ◽  
X Ray Crystallography ◽  
Automated Method

A fully automated method for detecting and centring protein crystals for X-ray crystallography is described. It relies on the analysis of rotating crystal trace images. No assumption is made concerning the shape, absolute size, or position of the crystal within its holder. Crystals as small as 15 µm were successfully positioned. This represents an important step towards high-throughput structural studies.

Biophysical applications in structural and molecular biology

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Solomon Tsegaye ◽  
Gobena Dedefo ◽  
Mohammed Mehdi
Keyword(s):  
Structural Biology ◽  
Structural Information ◽  
Conformational Dynamics ◽  
Biological Macromolecules ◽  
X Ray Diffraction ◽  
Biological Mechanisms ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Biophysical Techniques

Abstract The main objective of structural biology is to model proteins and other biological macromolecules and link the structural information to function and dynamics. The biological functions of protein molecules and nucleic acids are inherently dependent on their conformational dynamics. Imaging of individual molecules and their dynamic characteristics is an ample source of knowledge that brings new insights about mechanisms of action. The atomic-resolution structural information on most of the biomolecules has been solved by biophysical techniques; either by X-ray diffraction in single crystals or by nuclear magnetic resonance (NMR) spectroscopy in solution. Cryo-electron microscopy (cryo-EM) is emerging as a new tool for analysis of a larger macromolecule that couldn’t be solved by X-ray crystallography or NMR. Now a day’s low-resolution Cryo-EM is used in combination with either X-ray crystallography or NMR. The present review intends to provide updated information on applications like X-ray crystallography, cryo-EM and NMR which can be used independently and/or together in solving structures of biological macromolecules for our full comprehension of their biological mechanisms.

Structural studies of small amorphous volumes by electron diffraction

1990 ◽  
Vol 48 (4) ◽  
pp. 122-123
Author(s):  
Pierre Moine
Keyword(s):  
Electron Diffraction ◽  
Born Approximation ◽  
Structural Information ◽  
Fundamental Relation ◽  
X Rays ◽  
Structural Studies ◽  
Diffraction Experiment ◽  
Transmission Electron ◽  
Amorphous Structures ◽  
Diffraction Patterns

Qualitatively, amorphous structures can be easily revealed and differentiated from crystalline phases by their Transmission Electron Microscopy (TEM) images and their diffraction patterns (fig.1 and 2) but, for quantitative structural information, electron diffraction pattern intensity analyses are necessary. The parameters describing the structure of an amorphous specimen have been introduced in the context of scattering experiments which have been, so far, the most used techniques to obtain structural information in the form of statistical averages. When only small amorphous volumes (< 1/μm in size or thickness) are available, the much higher scattering of electrons (compared to neutrons or x rays) makes, despite its drawbacks, electron diffraction extremely valuable and often the only feasible technique.In a diffraction experiment, the intensity IN (Q) of a radiation, elastically scattered by N atoms of a sample, is measured and related to the atomic structure, using the fundamental relation (Born approximation) : IN(Q) = |FT[U(r)]|.

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