Macromolecular Crystallography Webinar training series
Welcome to the Rigaku Life Sciences Webinar Series. This free webinar series is designed to provide educational and training materials in X-ray crystallography methods. Each webinar will address a new topic ranging from optimization of crystallization conditions to solving the phase problem. We invite anyone to submit topic suggestions for future webinars and hope that you'll provide feedback for previous webinars.
Past webinars:
LS25: Tuesday, January 17, 2011
Recent Advances in Automated Protein Drop Imaging by Max Petersen
Analyzing vast numbers of protein drops is a time consuming bottleneck that affects most protein crystallization experiments. Automated imagers can reduce the time needed to inspect drops, shifting this bottleneck towards the analysis stage. The webinar gives a short overview on how automated imagers work and reviews techniques that can help reducing the time needed to analyze experiments. Focus is placed on discussion of specialized optics tailored to common drop dimensions, UV imaging, image processing techniques to quantify clear drops, as well as high resolution extended focus imaging.
LS24: Thursday, November 17, 2011
Home Lab SAD phasing with HKL-3000: From data collection to refined models in less than an hour by Jim Pflugrath
This interactive tutorial and webinar will demonstrate how to use HKL-3000 to process diffraction images, find the anomalous substructure with SHELXD, phase with SHELXC, MLPHARE, and DM, then build with ARP/wARP and refine with REFMAC (Kudos to all the authors of these programs!).
Emphasis will be on practical tips and how to interpret the output. Relatively low redundancy diffraction datasets will be used as examples to dispel some of the myths about sulfur and selenium Home Lab SAD phasing.
LS23: Thursday, September 1, 2011
An Insider's Guide to CrystalClear and d*TREK: Improve Your Results with these New Features and Tips by Jim Pflugrath
In his webinar Jim Pflugrath discussed three important new features or tips for using CrystalClear and d*TREK that will lead to better results: lower Rmeas and higher <I/σ(I)>. New input parameters for finding spots is one new feature. Another feature is a better way to perform positional refinement before and during Bragg reflection integration. A third tip is how to improve your scaling results by changing the input parameters to dtscaleaverage. These tips and others will give you better results with CrystalClear.
LS21: Thursday, June 23, 2011
Small Angle X-ray Scattering (SAXS) Techniques for Macromolecules by Angela Criswell
Small angle X-ray scattering is a technique used to study dilute solutions of macromolecules. SAXS measurements can give valuable information about the low resolution structural characteristics and in some cases a model of the protein shape. Specifically, SAXS is particularly useful for giving immediate feedback whether your protein is monodispersed or aggregated and whether your protein is folded or unfolded in solution. In addition, several examples in the literature demonstrate the use of SAXS to identify the correct multimeric states or proteins, thus eliminating the ambiguity associated with crystal-packing induced oligomers. This webinar will summarize various applications of SAXS for macromolecules and explore some practical considerations for collecting and processing SAXS data.
LS20: Thursday, May 12, 2011
SAD phasing at rotating anode wavelengths using iodide ions by Thomas E. Edwards
Phase determination remains a critical step in the determination of macromolecular structures using X-ray crystallography. When sufficient model phases cannot be generated via molecular replacement (MR), phases must be determined experimentally. For a large-scale structural genomics project, the Seattle Structural Genomics Center for Infectious Disease (SSGCID), we have adopted the use of iodide ion soaks and single-wavelength anomalous dispersion (SAD) experiments on data collected in-house using rotating anode X-ray generators or using tunable synchrotron beamlines as our primary method for de novo phase determination. This method uses existing native crystals and thus does not require laborious derivative-labeled protein preparation, crystallization re-optimization or synchrotron radiation. Iodide ions are non-toxic and soluble at molar concentrations, facilitating binding at numerous hydrophobic or positively charged sites, and have a strong anomalous signal in-house at copper or chromium rotating anode wavelengths. We have used this technique across a wide range of crystallization conditions with successful structure determination in at least twenty cases with a success rate of over 90% and recently reported our findings in the Journal of Structural and Functional Genomics In this webinar, we present a general overview of this method as well as several examples including SAD phasing and the combined use of SAD and MR for targets with weak MR solutions. These cases highlight the straightforward and powerful method of iodide ion SAD phasing.
LS19: Wednesday, February 23, 2011
Protein Crystallography: Getting in on the Ground Floor by Brian Matthews
Brian Matthews received both his B.S. (1959) and Ph.D. (1964) from the University of Adelaide, completing his studies during a pivotal period in X-ray crystallography—at around the same time that the first three-dimensional structure of a protein, myoglobin, was determined. "I had the good fortune to be a postdoc at the MRC Lab in Cambridge and to participate in the chymotrypsin structure determination in David Blow's group." In this webinar, Brian will share "a personal recollection of the structural biology at that time."
LS18: Wednesday, January 19, 2011
HKL-3000 - Toward the Future of Protein Crystallography by Wladek Minor
HKL-3000 integrates data collection, data reduction, phasing, and model building to significantly accelerate the process of structure determination, and on average, minimize the number of data sets and crystals required for structure solution. Execution of the package merges several modules and software applications into the structure determination pipeline. There are modules for experimental control of some beamlines and home instruments, data reduction, phasing by SAD/MAD or molecular replacement, fast model building, and initial refinement. The system is being developed and tested in the high-throughput environment of the Midwest Center for Structural Genomics (MCSG), Center for Structural Genomics of Infectious Diseases (CSGID) and New York Center for Structural Genomics (NYSGRC). The robustness of HKL-3000 has improved considerably over time and currently over 1500 structures have been determined with it.
The continuous advancement of the decision-making procedures within HKL-3000 have made it the system of choice for MCSG and CSGID projects. Transforming raw images into a solved structure (with 70% of the model built) in 10-15 minutes is no longer a surprise, but a routine operation for crystals that diffract to 2.5 Ǻ or better. Our experience with the determination of hundreds of structures by experimental phasing methods helped us to establish rules for the best approaches when the available data fall into three categories: unsolvable with current data, borderline and easy. Current work concentrates on improving the approach to borderline cases of structure determination rather than optimizing intermediate calculations for easy cases, thus shifting borderline cases into the easy category and unsolvable into borderline.
An important implication is that simple experimental protocols are sufficient in most cases and may even be optimal for the most challenging ones. Feedback from fast preliminary structure solution proved to be one of the critical components of success.
References
- Minor W, Cymborowski M, Otwinowski Z, Chruszcz M (2006) Acta Crystallographica Section D: Biological Crystallography 62: 859-66.
- Kirillova O, Chruszcz M, Shumilin IA, Skarina T, Gorodichtchenskaia E, Cymborowski M, Savchenko A, Edwards A, Minor W (2007) Acta Crystallographica Section D: Biological Crystallography 63: 348-54.
- Otwinowski Z, Borek D, Majewski W, Minor W (2003) Acta Crystallographica. Section A: Foundations of Crystallography 59: 228-34.
- Zheng H, Chruszcz M, Lasota P, Lebioda L, Minor W (2008) Journal of Inorganic Biochemistry 102(9): 1765-76.
- Chruszcz M, Wlodawer A, Minor W (2008) Biophysical Journal 95(1): 1-9.
- Wlodawer A, Minor W, Dauter Z, Jaskolski M (2008) Febs Journal 275: 1-21.
- Otwinowski Z, Minor W (1997) Methods in Enzymology 275: 307-326.
LS17: Thursday, November 18, 2010
When you need a challenge, try protein crystallisation (Tips and suggestions for helping you over the hurdles) by Janet Newman
Lots of people are interested in the information that one gets from seeing a picture of a protein in atomic detail. The most general way of getting this information is using X-ray diffraction techniques, which requires that the protein be coaxed into well-ordered crystals. This has become the bottleneck in structure determination. In this webinar Janet Newman discusses what you should know going into a crystallisation project, and offers suggestions about what to try, and what to expect from your screening steps, and also touches on some of the different things that one can try for optimising once you have an initial idea where your protein might like to crystallise. [Download slides]
LS16: Thursday, October 21, 2010
Scientific inquiry, inference and critical reasoning in the macromolecular crystallography curriculum by Bernhard Rupp
In this webinar, Bernhard Rupp expands on his recent Journal of Applied Crystallography article that discusses higher education curricula in the context of scientific analysis. Bernhard analyzes recent cases of high profile structure retractions and argues that "With the great power of modern crystallography comes great responsibility for its appropriate use." Specifically, Bernhard advocates that modern crystallographic curricula should emphasize hypothesis-driven testing and stress individual responsibility for published results. Drawing on postulates from Thomas Kuhn, Bernhard challenges us to re-evaluate how we engage the scientific method in both research and in educational settings.
LS15: Thursday, August 16, 2010
Structure Solution and Refinement with Phenix by Paul Adams
Phenix is a Python-based software system for the solution and refinement of crystallographic structures using X-ray and/or neutron diffraction data. Recent developments in Phenix, especially in the refinement program phenix.refine, are presented. These include real space target functions for refinement, the use of torsion angle constraints, and the application of additional restraints to improve structure refinement at low resolution. The integration of automated side chain rotamer correction into the refinement process will also be described.
LS14: Thursday, July 22, 2010
d*TREK & HKL-2000: Scaling and Statistics Revealed by Jim Pflugrath
This webinar spends more time on the scaling of Bragg reflections derived from diffraction images processed with d*TREK and other packages.
Examples of how to merge, scale, merge, and average data sets from the same crystal and/or from multiple crystals will be explained. Also shown will be how to use d*TREK to calculate your Table 1 for publication even if you use other packages to process and scale your data. The differences in the equations used by d*TREK and HKL-2000 will be highlighted and explained.
The final output can be used with the CCP4 or other packages to produce structural models.
LS13: Thursday, May 13, 2010
From Haemoglobin to West Nile Virus by Michael G. Rossmann
Fifty years ago, Max Perutz and John Kendrew at Cambridge University achieved something that many people at the time considered impossible: they were the first to use X-ray crystallography to decipher the molecular structures of proteins: haemoglobin and myoglobin. They found that both molecules were built from Linus Pauling's alpha helices, but folded and packed together in a complicated manner that never could have been deciphered by any other technique. With structure information in and they could then explain how haemoglobin in the bloodstream binds and releases oxygen on cue, how it passes its cargo on to the related storage protein myoglobin, and how a single amino acid mutation can produce the catastrophe known as sickle-cell anemia. Perutz and Kendrew also observed that the folding of helices was identical in myoglobin and the two chains of haemoglobin, and this along with the simultaneously evolving new technique of amino acid sequence analysis established for the first time the concept of molecular evolution.
The crystallographic puzzle was "cracked" by Perutz when he demonstrated that the binding of only two heavy metal atoms to horse haemoglobin changed the X-ray pattern enough to allow him to solve the "phase problem" and circumvent the main obstacle to protein crystal structure analysis. Because myoglobin has a single chain whereas haemoglobin has four, Kendrew's work with myoglobin progressed more rapidly; a low resolution structure appeared in 1956 and the high resolution structure in 1959. That same year saw the low resolution picture of haemoglobin, and the high resolution structure followed shortly thereafter.
Much of the work in structure analysis was carried out by visiting postdoctoral fellows and technicians, under the watchful eye of Perutz and Kendrew. Three of those former postdoctorals: Strandberg and Dickerson published a celebratory review for JMB in 2009. In this webinar Michael G. Rossmann provided a personal account of these events.
LS12: Thursday, April 22, 2010
Diffraction Image Processing with d*TREK by Jim Pflugrath
This webinar demonstrates how to use the d*TREK suite of programs to process single crystal X-ray diffraction images from a variety of crystals and detectors. Jim Pflugrath presents first the quick automated method, followed by instructions on finding the X-ray beam center, masking shadows on the detector, refining your goniometer imperfections, scaling the resultant integrated measurements, and determining the crystal space group. He also covers some advanced d*TREK usage including scripting, ranking of crystals, and processing multiple scans.
LS11: Thursday, March 18, 2010
Processing diffraction data with imosflm by Andrew Leslie
Andrew Leslie demonstrated the use of the imosflm interface to process X-ray diffraction images. imosflm is part of the CCP4 package, and provides a user-friendly but powerful interface to the mosflm program. Hints on how to interpret the program output, how to get the best out of the package, and how to deal with challenging cases were provided. Andrew works at the MRC Laboratory of Molecular Biology in Cambridge, UK.
LS10: Thursday, February 25, 2010
Practical Approaches to Data Processing Using XDS
In this webinar, Kay Diederichs provided an overview of X-ray diffraction image processing with XDS. Kay created the XDSwiki in 2008 to provide an accurate and up-to-date resource for XDS users. He contributes to the development of XDS and is the author of XDSSTAT, a program that provides additional analytical tools for XDS output. Kay is also the originator of the CCP4wiki and a frequent contributor to the CCP4bb. He is currently a Professor at Universität Konstanz.
LS9: November 19, 2009
Practical Aspects of SAD Data Collection
In this webinar, John Rose covered Single wavelength Anomalous Diffraction enhanced by chromium radiation (CrSAD). Prof. Rose reviewed the reasons for using SAD for de novo and phased molecular replacement structure solution and described the method. Prof. Rose then discussed the practical details of using SAD and walked through an example of sulfur SAD phasing using zinc-free insulin.
LS8: October 28, 2009
High-Throughput Structural Biology at the JCSG
In this webinar, Ian Wilson describes the current state of High-Throughput Structural Biology at the Joint Center for Structural Genomics. The JCSG is funded by the National Institute of General Medical Sciences (NIGMS), as part of the second phase of the Protein Structure Initiative (PSI) of the National Institutes of Health. (U54 GM074898).
The JCSG is committed to a model that employs distributed local management and centralized coordination to establish a pipeline with fully functional large-scale production sites that are tightly integrated to ensure standardization of data, uniformity of communication interfaces, and synchronization of production schedules. The overall structure of the JCSG is designed to ensure the most efficient operation and data management that optimizes the use of resources and minimizes the cost per structure. This "factory"-like approach was developed in a scalable mode to enable the JCSG to easily adapt to changing goals and challenges, which importantly includes a learning environment to feed results back from prior experiments in order to increase productivity at each step in the process.
The JCSG is a multi-institutional consortium with major activities at The Scripps Research Institute (TSRI); the Genomics Institute of the Novartis Research Foundation (GNF); the University of California, San Diego (UCSD); the Burnham Institute for Medical Research (Burnham); and the Stanford Synchrotron Radiation Lightsource (SSRL) at Stanford University.
LS7: September 30, 2009
Small angle X-ray scattering techniques for proteins
In this webinar we explore the application of small angle X-ray scattering (SAXS) techniques to the study of non-crystalline macromolecular samples in solution. SAXS measurements have shown to be powerful for both ab initio modeling where the structure of the protein is unknown, and fragment based rigid body modeling where some structural information may already be available. Easy control of sample conditions allows SAXS measurements under conditions similar to their physiological environment. SAXS measurements can also be useful to follow the response of samples to perturbations of temperature, compound addition, and other changes to the physical or chemical environment. The webinar includes an overview of the theory and techniques associated with SAXS measurements, experimental hardware for the home laboratory, basic data processing, examples of laboratory based SAXS measurements and comparison to measurements made using synchrotron radiation. Download this presentation
LS6: August 26, 2009
Caveat Emptor: What is the Right X-ray Source for You?
In the previous webinar in this series, we reviewed some of the maintenance procedures associated with rotating anode X-ray generators. Now Joe Ferrara explores the properties of X-ray sources; that is, the combination of X-ray generator and X-ray optic. We cover properties such as focal spot size, divergence, spectral purity, beam size and shape and how they affect data quality. Upon completion you will have a better understanding of what type of source best suits your needs.
LS5: July 16, 2009
Maintenance for rotating anode X-ray generators
Modern microfocus rotating anode generators like the MicroMax-007 and MicroMax-007 HF provide higher performance but also require less maintenance and service than older generators. This webinar, presented by RIgaku analytical X-ray systems specialist Adam Courville, will review the maintenance and service required for a MicroMax-007 series generator and present best practices and tips on how to keep your MicroMax-007 generator working at its best.
LS4: June 24, 2009
How to collect exceptional diffraction data for your crystals
With modernized generators, detectors, remote capabilities, and robots it seems that more and more users treat the diffraction experiment as a black box... "Shoot first and ask questions later." Fortunately, this strategy works in some cases but in others a bad data set can often lead to bad results during the structure solution and refinement process. This webinar, presented by Angela Criswell, describes the diffraction experiment in terms of best practices and provide tools both for identifying diffraction quality samples and for collecting exceptional diffraction data.
LS3: May 27, 2009
X-ray radiation safety — What everyone should know
Modern X-ray diffraction systems are more powerful than their predecessors with regard to raw beam intensities. As a result, it is more important than ever for users to have a strong understanding of the risks of radiation exposure, the biological results of exposures, and ways to limit the risk. In this webinar, Kris Tesh will present information about X-ray radiation safety and what every user should know.
LS2: Apr 23, 2009
Getting Funded: What's Hot and What's Not
The stimulus package brought forth by President Obama has made for exciting times at NIH and other federal funding organizations. While it may be easier than ever before to get funding in the life sciences you still need to make sure your grant proposal meets the requirements of the funding agency. Your proposal must also provide the appropriate background information for your particular research field, your contribution to that field , a detailed discussion of your proposal's broader impact, and why your proposal is worthy of funding based on purely intellectual merit. We will review both general tip and tricks for all proposals as well as some specific tips and tricks for proposals to NIH.
LS1: Mar 31, 2009
Macromolecular cryo-crystallography: some opinions about best practices
Jim Pflugrath discussed techniques and best practices for cryo-crystallography some of which can be found in a review paper published in 2004, Macromolecular cryocrystallography—methods for cooling and mounting protein crystals at cryogenic temperatures.
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