scholarly journals Preventing Bio-Bloopers and XFEL Follies: Best Practices from your Friendly Instrument Staff

Crystals ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 251 ◽  
Author(s):  
Christopher Kupitz ◽  
Raymond G. Sierra
Keyword(s):  
Best Practices ◽  
Structural Biology ◽  
High Speed ◽  
X Ray ◽  
Experimental Planning ◽  
The Past ◽  
Delivery Technique ◽  
Scientific Goal ◽  
Paradigm Shifting ◽  
Serial Femtosecond Crystallography

Serial Femtosecond Crystallography (SFX) at X-ray Free electron Lasers (XFELs) is a relatively new field promising to deliver unparalleled spatial and temporal resolution on biological systems and there dynamics. Over the past decade, though, there have been a handful of results that have truly delivered on these promises. Why? SFX has many paradigm shifting techniques not seen in typical structural biology arenas, such as creating a concentrated slurry of microcrystals rather than a handful of loopable prisms worthy of a catalog photo. Then taking that slurry and high speed jetting them towards the vacuum or helium interation region to destroy less than 1% of your sample and waste the other 99. The literature is full of techniques and methods promising to cure what ails your experiment, yet as an instrument scientist will tell you –and a first author might admit after a few drinks at the conference happy hour—is that there are a lot more failures than the success we published, results may vary. We will walk through a best practices on how to prepare your sample and chose a sample delivery technique that will amerliorate your studies rather than undermine your hardwork and hopefully lead to better experimental planning and execution, inching you closer to that scientific goal and that call from Stockholm. This will be written in a more editorialized fashion and is meant to give the reader an idea of what to try or how they should be thinking. Welcome to SFX, now what?

scholarly journals Discerning best practices in XFEL-based biological crystallography – standards for nonstandard experiments

IUCrJ ◽  
2021 ◽  
Vol 8 (4) ◽  
pp. 532-543
Author(s):  
Alexander Gorel ◽  
Ilme Schlichting ◽  
Thomas R. M. Barends
Keyword(s):  
Best Practices ◽  
Structural Biology ◽  
Conformational Changes ◽  
Critical Evaluation ◽  
Quality Standards ◽  
Free Electron Lasers ◽  
High Resolution Data ◽  
X Ray ◽  
Serial Femtosecond Crystallography ◽  
Resolution Data

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a novel tool in structural biology. In contrast to conventional crystallography, SFX relies on merging partial intensities acquired with X-ray beams of often randomly fluctuating properties from a very large number of still diffraction images of generally randomly oriented microcrystals. For this reason, and possibly due to limitations of the still evolving data-analysis programs, XFEL-derived SFX data are typically of a lower quality than `standard' crystallographic data. In contrast with this, the studies performed at XFELs often aim to investigate issues that require precise high-resolution data, for example to determine structures of intermediates at low occupancy, which often display very small conformational changes. This is a potentially dangerous combination and underscores the need for a critical evaluation of procedures including data-quality standards in XFEL-based structural biology. Here, such concerns are addressed.

New developments in the theory and interpretation of X-ray spectra based on fast parallel calculations

2002 ◽  
Vol 10 (1) ◽  
pp. 43-45 ◽  
Author(s):  
J. J. Rehr ◽  
A. L. Ankudinov
Keyword(s):  
Ab Initio ◽  
Structural Biology ◽  
Significant Progress ◽  
Edge Structure ◽  
New Developments ◽  
X Ray ◽  
The Past ◽  
Current State ◽  
X Ray Absorption ◽  
Made In

There has been dramatic progress over the past decade both in theory and inab initiocalculations of X-ray absorption fine structure. Significant progress has also been made in understanding X-ray absorption near-edge structure (XANES). This contribution briefly reviews the developments in this field leading up to the current state. One of the key advances has been the development of severalab initiocodes such asFEFF, which permit an interpretation of the spectra in terms of geometrical and electronic properties of a material. Despite this progress, XANES calculations have remained challenging both to compute and to interpret. However, recent advances based on parallel Lanczos multiple-scattering algorithms have led to speed increases of typically two orders of magnitude, making fast calculations practicable. Improvements in the interpretation of near-edge structure have also been made. It is suggested that these developments can be advantageous in structural biology,e.g.in post-genomics studies of metalloproteins.

scholarly journals In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology?

2014 ◽  
Vol 369 (1647) ◽  
pp. 20130497 ◽  
Author(s):  
François-Xavier Gallat ◽  
Naohiro Matsugaki ◽  
Nathan P. Coussens ◽  
Koichiro J. Yagi ◽  
Marion Boudes ◽  
...  
Keyword(s):  
Structural Biology ◽  
Free Electron ◽  
Free Electron Laser ◽  
Protein Crystal ◽  
X Ray ◽  
Sample Characterization ◽  
The One ◽  
Serial Femtosecond Crystallography

The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography.

scholarly journals Structural Biology of Influenza Hemagglutinin: An Amaranthine Adventure

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1053
Author(s):  
Nicholas C. Wu ◽  
Ian A. Wilson
Keyword(s):  
Membrane Fusion ◽  
Structural Biology ◽  
Receptor Binding ◽  
Viral Entry ◽  
Antigenic Drift ◽  
Current Status ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Past ◽  
Influenza Hemagglutinin

Hemagglutinin (HA) glycoprotein is an important focus of influenza research due to its role in antigenic drift and shift, as well as its receptor binding and membrane fusion functions, which are indispensable for viral entry. Over the past four decades, X-ray crystallography has greatly facilitated our understanding of HA receptor binding, membrane fusion, and antigenicity. The recent advances in cryo-EM have further deepened our comprehension of HA biology. Since influenza HA constantly evolves in natural circulating strains, there are always new questions to be answered. The incessant accumulation of knowledge on the structural biology of HA over several decades has also facilitated the design and development of novel therapeutics and vaccines. This review describes the current status of the field of HA structural biology, how we got here, and what the next steps might be.

scholarly journals The Diamond Light Source and the challenges ahead for structural biology: some informal remarks

2015 ◽  
Vol 373 (2036) ◽  
pp. 20130156 ◽  
Author(s):  
V. Ramakrishnan
Keyword(s):  
Synchrotron Radiation ◽  
Light Source ◽  
Structural Biology ◽  
Complex Structures ◽  
Biological Processes ◽  
Short Article ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Past

The remarkable advances in structural biology in the past three decades have led to the determination of increasingly complex structures that lie at the heart of many important biological processes. Many of these advances have been made possible by the use of X-ray crystallography using synchrotron radiation. In this short article, some of the challenges and prospects that lie ahead will be summarized.

scholarly journals Two-dimensional imaging detectors for structural biology with X-ray lasers

2014 ◽  
Vol 369 (1647) ◽  
pp. 20130334 ◽  
Author(s):  
Peter Denes
Keyword(s):  
Structural Biology ◽  
Peak Power ◽  
State Of The Art ◽  
High Peak Power ◽  
Free Electron Lasers ◽  
X Ray ◽  
The Past ◽  
Current State ◽  
Pixel Detectors ◽  
Imaging Detectors

Our ability to harness the advances in microelectronics over the past decade(s) for X-ray detection has resulted in significant improvements in the state of the art. Biology with X-ray free-electron lasers present daunting detector challenges: all of the photons arrive at the same time, and individual high peak power pulses must be read out shot-by-shot. Direct X-ray detection in silicon pixel detectors—monolithic or hybrid—are the standard for XFELs today. For structural biology, improvements are needed for today's 10–100 Hz XFELs, and further improvements are required for tomorrow's 10+ kHz XFELs. This article will discuss detector challenges, why they arise and ways to overcome them, along with the current state of the art.

scholarly journals LA RIVOLUZIONE DELLA CRIO-MICROSCOPIA ELETTRONICA NELLO STUDIO STRUTTURALE DI PROTEINE E ACIDI NUCLEICI

2020 ◽  
Author(s):  
Martino Bolognesi
Keyword(s):  
Electron Microscopy ◽  
Amino Acids ◽  
Structural Biology ◽  
Molecular Structures ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Past ◽  
Cryo Electron Microscopy ◽  
Resolution Level ◽  
Nello Studio

Observing the fine details of molecular structures (e.g. in proteins and in nucleic acids) has been a central part of Structural Biology over the past 50 years. The recent advent of single particle cryo-electron microscopy brought a revolution in this field, that previously relied on X-ray crystallography and nuclear magnetic resonance. It is now possible to explore the structures of large subcellular assemblies, such as the ribosome, resolving details on the scale of amino acids and nucleotides, in favorable cases reaching the 2 Å resolution level.

scholarly journals Microcrystallization techniques for serial femtosecond crystallography using photosystem II from Thermosynechococcus elongatus as a model system

2014 ◽  
Vol 369 (1647) ◽  
pp. 20130316 ◽  
Author(s):  
Christopher Kupitz ◽  
Ingo Grotjohann ◽  
Chelsie E. Conrad ◽  
Shatabdi Roy-Chowdhury ◽  
Raimund Fromme ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
High Energy ◽  
Model System ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
Membrane Protein Complex ◽  
X Ray ◽  
Free Interface ◽  
The Past ◽  
Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) is a new emerging method, where X-ray diffraction data are collected from a fully hydrated stream of nano- or microcrystals of biomolecules in their mother liquor using high-energy, X-ray free-electron lasers. The success of SFX experiments strongly depends on the ability to grow large amounts of well-ordered nano/microcrystals of homogeneous size distribution. While methods to grow large single crystals have been extensively explored in the past, method developments to grow nano/microcrystals in sufficient amounts for SFX experiments are still in their infancy. Here, we describe and compare three methods (batch, free interface diffusion (FID) and FID centrifugation) for growth of nano/microcrystals for time-resolved SFX experiments using the large membrane protein complex photosystem II as a model system.

The Use of Advanced Analytical Techniques for Characterization of the Chemical, Physical, and Crystallographic Nature of a Surface

1973 ◽  
Vol 31 ◽  
pp. 132-133 ◽  
Author(s):  
R. E. Herfert
Keyword(s):  
Surface Characterization ◽  
Analytical Techniques ◽  
X Ray Diffraction ◽  
Depth Of Penetration ◽  
X Ray ◽  
The Past ◽  
Transmission Electron ◽  
Scanning Electron

Studies of the nature of a surface, either metallic or nonmetallic, in the past, have been limited to the instrumentation available for these measurements. In the past, optical microscopy, replica transmission electron microscopy, electron or X-ray diffraction and optical or X-ray spectroscopy have provided the means of surface characterization. Actually, some of these techniques are not purely surface; the depth of penetration may be a few thousands of an inch. Within the last five years, instrumentation has been made available which now makes it practical for use to study the outer few 100A of layers and characterize it completely from a chemical, physical, and crystallographic standpoint. The scanning electron microscope (SEM) provides a means of viewing the surface of a material in situ to magnifications as high as 250,000X.

RNA polymerase: Preparation and structural analysis of helical polymers

1984 ◽  
Vol 42 ◽  
pp. 152-153
Author(s):  
E. Loren Buhle ◽  
Pamela Rew ◽  
Ueli Aebi
Keyword(s):  
Structural Analysis ◽  
Rna Polymerase ◽  
Molecular Level ◽  
X Ray Diffraction ◽  
Helical Polymers ◽  
X Ray ◽  
E Coli ◽  
The Past ◽  
Ordered Arrays ◽  
Low Ionic Strength

While DNA-dependent RNA polymerase represents one of the key enzymes involved in transcription and ultimately in gene expression in procaryotic and eucaryotic cells, little progress has been made towards elucidation of its 3-D structure at the molecular level over the past few years. This is mainly because to date no 3-D crystals suitable for X-ray diffraction analysis have been obtained with this rather large (MW ~500 kd) multi-subunit (α2ββ'ζ). As an alternative, we have been trying to form ordered arrays of RNA polymerase from E. coli suitable for structural analysis in the electron microscope combined with image processing. Here we report about helical polymers induced from holoenzyme (α2ββ'ζ) at low ionic strength with 5-7 mM MnCl2 (see Fig. 1a). The presence of the ζ-subunit (MW 86 kd) is required to form these polymers, since the core enzyme (α2ββ') does fail to assemble into such structures under these conditions.

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