July - August 2011

New from MiTeGen

• Small Molecule Starter Kits

Customer Contributions

• The Importance of Crystal Mounts
• Poster Prize: Transition Metal Complexes for the Reduction of Carbon Dioxide

Product Highlights

• Jena : JBScreen Pentaerythritol
• MicroLoops LD™
• CryoVial, Base and Crystal Mount Assemblies

Other News

• Recent Press releases
• Monthly Tech Tip
• Recent Citations

Welcome

Welcome to the July - August Mitegen Newsletter.

In this issue Simon Coles of the UK National Crystallographic Service shares his experience when switching to Mitegen Mounts, and we introduce our new Starter Kits for small molecule crystallographers.

We also extend our congratulations to Timothy Schmeier of Yale for winning the Mitegen poster prize at the 3rd annual Yale-Rigaku Symposium 2011 that took place in June.

Of course, we look forward to seeing many of you at the upcoming XXII International Congress - IUCR2011 in Madrid in late August. Visit us there at booth 55 to see our products and discuss your applications and challenges.

Robert Newman
CEO
Mitegen

Customer feedback on our products and newsletter is always welcome.

New from MiTeGen

Small Molecule Starter Kits

Mitegen's Small Molecule Crystallography Starter Kit has everything you need to mount and collect X-ray data from your small molecules at both room temperature and at cryo temperatures.

The SMSK-1 kit contains:

• 20 Dual Thickness MicroMounts for Small Molecules (M2-L18SP-A2-SM), with 5 each of 75, 100, 150 and 200 μm apertures
• 10 GB-B3S-R reusable goniometer bases
• 10 magnetic CryoVials
• 1 tube of Apiezon N cryogenic vacuum grease
• 1 pair heavy-duty tweezers, for inserting the MicroMounts into the bases
• A detailed instruction manual

Customer Contribution

The Importance of Crystal Mounts

By: Simon Coles
Director, UK National Crystallography Service
School of Chemistry,
University of Southampton.

At the UK National Crystallography Service our work is mainly concerned with looking at the samples that other crystallographers cannot handle with their regular diffraction facilities. This means we spend a huge amount of time looking at very small and weakly diffracting samples. It goes without saying that we need very intense x-ray sources for this and accordingly we have been pioneering the use of rotating anodes and synchrotron sources for chemical crystallography for some time now.

The first time I really understood the importance of crystal mounts for this type of work was when building the worlds first small molecule single crystal diffraction beamline – 9.8 at Daresbury. Back then, in order to minimize low angle scatter by the mount, we devised a two stage system which consisted of gluing a strand of glass wool to a drawn out capillary – this worked pretty well, but was extremely tedious to make and very prone to breaking! Our attention to crystal mounting was revisited around 2004 when we were developing the first focusing optics for Mo radiation in our home lab – we soon realized that a big jump in intensity meant a significant increase in background scatter from the mount!

I will always remember one of the team at the time coming back from a conference with one of the early 'Mitegen style' mounts – he burst into my office saying hey look at this its like a fountain pen nib for crystal mounting. We were immediately sold on the idea – not only is a Mitegen mount considerably more transparent to x-rays than glass (!) but its unique design means there is generally less 'foreign' material in the beam. Using Mitegen's in our work has transformed the quality of the data we get – especially when we have had to do extensive crystal cleaning - a process we often have to perform in a pool of oil. However, most certainly the biggest impact Mitegen mounts have made on our work is in the handling of extremely sensitive crystals – again these need to be manipulated under oil or grease to avoid them decomposing through exposure to air or by loss of solvent of crystallization and Mitegen mounts really reduce the amount of low angle background scatter and thereby significantly increase the quality of data we get from these very challenging samples.

About the UK National Crystallography Service

The Service is an amalgamation of resources at two centres; laboratory-based facilities in the Chemical Crystallography Laboratory at the School of Chemistry, University of Southampton, together with provision of a synchrotron-based facility on station I19 at the Diamond Light Source. For more information got to http://www.ncs.ac.uk/

Customer Quote:

In short, I would strongly advise all small molecule laboratories to move over to using Mitegen mounts – you really will find it easier to manipulate your crystals and you will certainly notice the increase in data quality!"

Simon Coles
Director, UL National Crystallographic Service School of Chemistry
University of Southampton

Jena Bioscience product highlight

JBScreen Pentaerythritol has been designed for efficient crystallization screening of biological macromolecules based on pentaerythritol polymers as precipitants. The screen was developed by Ulrike Demmer from the Max-Planck-Institute for Biophysics in Frankfurt. The choice of a suitable precipitant is of crucial importance for the crystallization of proteins. JBScreen Pentaerythritol utilizes two novel precipitating agents, i.e. pentaerythritol propoxylate and pentaerythritol epoxylate. Both are branched polymers containing a pentaerythritol backbone. Thus they differ from more traditional precipitants like MPD and PEG’s in size and nature.

In addition, pentaerythritol polymers function as cryoprotectants. Protein crystals grown in high concentrations of these precipitants can be frozen directly from the crystallization drop. The successful application of pentaerythritol polymers to yield protein crystals was first described by Gulick et al. [1]. Now this class of precipitants has been used for membrane crystallization, too. The X-ray structure of cbb3 Cytochrome Oxidase was recently published in Science [2]. Crystals of this proton pumping membrane protein where successfully grown using pentaerythritol ethoxylate as precipitation agent.

JBScreen Pentaerythritol comprises of 96 unique conditions, based on 4 different pentaerythritol polymers as precipitating agent:

• Pentaerythritol propoxylate 426 (5/4 PO/OH)
• Pentaerythritol propoxylate 629 (17/8 PO/OH)
• Pentaerythritol ethoxylate 270 (3/4 EO/OH)
• Pentaerythritol ethoxylate 797 (15/4 EO/OH)

The 4 polymers are arranged to a grid screen, thus allowing screening i) of three different precipitant concentrations, ii) four different pH values and iii) with and without the addition of salts, i.e. magnesium chloride, ammonium sulfate, potassium chloride.

The advantage of JBScreen Pentaerythritol not only lies in the novel 96 conditions but also in the systematic arrangement of the unique reagents, which enables the user to compare individual conditions directly. Even if initial screening may not always yield crystals, valuable information about the protein under investigation can be obtained from the scoring sheet. JBScreen Pentaerythritol Scoring Sheet

All JBScreen Pentaerythritol screening kits include a detailed production report and data sheets.

Individual Conditions of all screens are also available in 10 ml as well as 100 ml volumes.

Recent Press Releases

Spotlight:
Timothy Schmeier of Yale University recieves Mitegen poster prize at the 2011 Yale-Rigaku Symposium

Timothy Schmeier of Yale University has been awarded the Mitegen poster prize in recognition of excellent research in the area of X-ray crystallography for his poster titled “Transition Metal Complexes for the Reduction of Carbon Dioxide.” The poster was presented at the 3rd Annual Yale-Rigaku Symposium which explores recent advances in research fields associated with X-ray crystallography.

The organometallic chemistry laboratory supervised by Professor Nilay Hazari at Yale University investigates the insertion carbon dioxide into various metal-hydride, metal-alkyl, and metal-heteroatom bonds (M-R) to form the respective metal-carboxylates (M-OC(O)R). The application of this fundamental research is then used to design new catalysts for the reduction of carbon dioxide into commodity chemicals.

Recent work presented by Timothy Schmeier included a highly active PNP pincer-supported iridium trihydride catalyst which reduces carbon dioxide to potassium formate at turn over numbers exceeding 345,000¹. X-ray crystallography was shown to be vital to this research as it revealed a secondary coordination sphere hydrogen bond present in the carbon dioxide insertion product, an iridium formate complex, which could not be detected with any spectroscopic methods. The hydrogen bond detected between the NH bond present in the pincer ligand and the metal-formate group is hypothesized to facilitate the insertion of carbon dioxide into the iridium-hydride bond and also stabilize the resulting iridium-formate complex.

1. Schmeier, T.J. et al. Secondary Coordination Sphere Interactions Facilitate the Insertion Step in an Iridium(III) CO(2) Reduction Catalyst.  Journal of the American Chemical Society 133, 9274-7(2011).

Customer Quote:

"Mitegen's RT system is very handy and useful for room temperature measurements. It makes life easy in handling crystals at room temperature. We have been extensively using it for our room temperature studies. It was also very helpful in dehydration studies. In fact we also use Mitegen meshes for handling our micro crystals, which is excellent.

We are very impressed by the constant innovative design and improvement of the Mitegen products which is very helpful and caters to the needs of the users"

Rajendran Chitra
MIX Operator
Macromolecular Crystallography
Swiss Light Source
Paul Scherrer Institute (PSI)

Monthly Tech Tip

Reducing the Mosaicity of Flash-Cooled Crystals

Flash cooling protein and virus crystals always increases their mosaicity. As-grown, room-temperature mosaicities are often as small as 0.005° (when measured using small-divergence X-ray sources). But mosaicities at T=100 K are typically between 0.3° and 1° . Large mosaicities reduce diffraction signal to noise (especially when low-divergence X-ray beams are used) and increase spot overlap. What can you do to reduce flash-cooling-related mosaicity increase?

First, verify that cooling your crystals is the source of your poor mosaicity. Make sure that the crystal isn’t visibly moving in the cryostream, when viewed using a high magnification telescope or camera. Then check the diffraction of your crystal before you freeze it using the MicroRT system. Use short exposures to minimize radiation damage.

Assuming your room temperature mosaicity is ok, here are some things to try:

• Reduce the amount of excess liquid around your crystal. When this liquid freezes, it exerts stresses on the crystal that can bend or crack it. Use a mount with an aperture that’s a bit smaller than the crystal. Carefully remove excess liquid using a size 15 paper wick or by gently tapping on the steel rod of the mount..
• If possible, avoid using thin plates and rods, which are more easily bent and cracked. Smaller, more three dimensional crystals can give better results.
• Reduce the beam spot size to examine only a small part of the sample, and scan the beam relative to the sample to find the region with the lowest mosaicity.
• Make sure that the crystal isn’t dehydrating just before or during flash cooling. This is especially important for smaller crystals, which dry out very quickly. Transfer the crystal to LV Cryo Oil before mounting, and mount with as little remaining oil as possible.
• Look carefully at your sample through the telescope or on the video monitor. If it’s fluttering in the cryostream, follow the instructions here to reduce sample motion.
• Make sure your flash cooling set-up and protocol is giving fast and uniform cooling. Warkentin et al.'s hyperquenching protocol (J. Appl. Cryst. (2006) 39, 805-811) , a variant on standard plunge cooling in liquid nitrogen in which you you blow away the cold gas layer that forms above the liquid nitrogen, gives the most reliable results.
• If fast cooling doesn’t succeed, try slowly cooling the crystal using the protocol of Warkentin et al. (J. Appl. Cryst. (2009) 42).

Contact Us with comments or suggestions

Customer Quote:

"Mitegen MicroMounts are excellent.

Its simple yet elegant design allows scooping up crystals easily.

I usually have my samples in a syrupy oil and grab the right size crystals with a Mitegen.

I like the perforation and engravings on the Mitegens as they let me visualize the crystal size."

Navamoney Arulsamy
Research Scientist
Department of Chemistry
University of Wyoming

Select Recent Citations

Makoto Tanabe, Masaya Hanzawa & Kohtaro Osakada (2011) Ge–Ge Bond-Forming Reactions from Bis(germyl)palladium Complexes with Chelating Diphosphine Ligands  Phosphorus, Sulfur, and Silicon and the Related Elements, Volume 186, Issue 6, 2011, pages 1384-1388

Takashi Wadaa, Kazumi Shimono, Takashi Kikukawa, Masakatsu Hato, Naoko Shinya, So Young Kim, Tomomi Kimura-Someya, Mikako Shirouzu, Jun Tamogami, Seiji Miyauchi, Kwang-Hwan Jung, Naoki Kamo, and Shigeyuki Yokoyama (2011) Crystal structure of the eukaryotic light-driven proton pumping rhodopsin, Acetabularia rhodopsin II, from marine alga  Journal of Molecular Biology

J. Porta, J. J. Lovelace, A. M. M. Schreurs, L. M. J. Kroon-Batenburg and G. E. O. Borgstahl (2011) Processing incommensurately modulated protein diffraction data with Eval15  Acta Crystallographica Section D, Volume 67, Part 7 (July 2011)

Loi H. Do and Stephen J. Lippard (2011) Toward Functional Carboxylate-Bridged Diiron Protein Mimics: Achieving Structural Stability and Conformational Flexibility Using a Macrocylic Ligand Framework  Journal of the American Chemical Society,

Lorenz Michael Reith, Martin Stiftinger, Uwe Monkowius, Gnther Knr, and Wolfgang Schoefberger (2011) Synthesis and Characterization of a Stable Bismuth(III) A₃–Corrole  Inorganic Chemistry, June 13, 2011

A. S. M. Soliman, M. Warkentin, B. Apker and R. E. Thorne (2011) Development of high-performance X-ray transparent crystallization plates for in situ protein crystal screening and analysis   Acta Crystallographica Section D, Volume 67, Part 7 (July 2011)

Every month, the use on Mitegen products is cited in dozens of papers.
For more citations, click here.

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