September - October 2011

circulation this issue: 6,896

New from MiTeGen

• MicroCrystal Mounts

Customer Contributions

• The benefits of MicroLoops E for mounting needle-shaped crystals
• Spotlight: Dr. Shane Telfer, Institute of Fundamental Sciences, Massey University

Product Highlights

• Jena : Phasing Kits
• MicroLoops E™ for Needle and rod shaped crystals
• Micro RT™ and reusable bases
• Quick Puck Loader
• MicroLoops LD™

Other News

• Recent Press releases
• Monthly Tech Tip
• Recent Citations

Welcome

Welcome to the September - October Mitegen Newsletter.

We'd like to thank the hundreds of customers that stopped by our booth at the IUCr meeting in Madrid last month, making the event a great success for Mitegen.

This month we are highlighting MicroLoops E for needle and rod shape crystals, discussed in the contibution from Monika-Sarah Schulze at the
Dana-Farber Cancer Institute, and also in our tech tips. 

Also this month, Dr. Shane Telfer of Massey University discusses his studies of metal-organic frameworks.

New tools being introduced include MicroCrystal Mounts, and a Quick Puck Loader for error free loading of Rigaku pucks.

Robert Newman
CEO
Mitegen

Customer feedback on our products and newsletter is always welcome.

* new: follow us on facebook:

New from MiTeGen

MicroCrystal Mounts

Specifically designed for:

• Crystals smaller than 20 μm
• Crystallography on micro-focus sources
• Ultra-low background scatter and superior crystal visualization

These ultra-transparent mounts meet the challenges of visualizing, aligning, and collecting diffraction data from micron size crystals. Using dual-thickness technology, crystals are supported on a 3 micron thick film in a 10 micron thick frame. An aerodynamic design combined with a shorter tip length minimizes sample motion. MicroCrystal Mounts provide an a unsurpassed combination of X-Ray transparency and rigidity.

Customer Contribution

The benefits of MicroLoops E for mounting needle-shaped crystals

By: Monika-Sarah Schulze
PhD candidate
Dana-Farber Cancer Institute
Harvard Medical School

Our group is working on MHC II (major histocompatibility complex class II) molecules which play a crucial role in initiating immune responses to pathogens. MHC II molecules are surface proteins which present antigenic peptides to T cells and we are interested in the process of how MHC II molecules are loaded with antigens. Last year I obtained very nice looking crystals of a MHC II protein but unfortunately due to large unit cell dimensions, pseudo-translational symmetry and potential twinning, it was difficult to refine the protein structure.

This year I went to a CCP4 workshop at the Advanced Photon Source close to Chicago. Beforehand I had obtained a new crystal form and I brought several crystals to the workshop for measuring at the beamline of GM/CA-CAT. I was not very optimistic about the new protein crystals as they were needle-shaped, quite fragile and difficult to handle. I probably would have tried to mount the crystals using round nylon loops which previously had caused a lot of damage to and breakage of the crystalline needles. But fortunately, MiTeGen provided samples for the course and sent me differently sized MicroLoops E which feature an elongated thin polymer aperture specifically designed for needle- and rod-shaped crystals. The MicroLoops were already attached to ALS-style goniometer bases, which I could then use with the automounter at GM/CA-CAT.

For me it made a big difference to use the uniquely designed apertures during needle mounting compared to conventional loops. First I found it very easy to place the flat and elongated aperture beneath the crystal and I could even detach some needles from the glass cover by gently sliding the long and bending tip under the needle. Picking up the crystal was also facilitated and accompanied by less crystal damage because the fingers extending into the aperture formed a supporting base similar to a mesh, which also prevented crystal slippage. Although I was first worried that the intensive contact between needle and aperture would cause crystal cracks upon flash-cooling, I observed no evidence of damage. I was also pleasantly surprised at how little liquid remained around the needle after mounting (see image), minimizing background scatter.

Overall using MicroLoops E, I managed to freeze longer intact needles (~ 600 um x 50 um) than I could have with conventional loops. As radiation damage was a problem, having longer needles mounted was definitely important for data collection because a larger crystal volume could be used for stepwise X-ray exposure. In the end, I successfully collected several complete data sets from individual crystals which diffracted to around 2.2 Angstrom and showed no indication of pseudo-translation symmetry or twinning.

About Monika-Sarah Schulze:

Monika-Sarah Schulze graduated from the University of Freiburg, Germany. During her Master’s thesis she worked in the protein crystallography group of Dr. Georg E. Schulz investigating general principles of protein-protein interactions. Currently she is in the lab of Dr. Kai Wucherpfennig in the Department of Cancer Immunology and AIDS at Dana-Farber Cancer Institute, which is affiliated with Harvard Medical School. For her PhD thesis she is studying the mechanism of peptide loading onto MHC II molecules, applying functional assays such as surface plasmon resonance as well as structural methods including X-ray crystallography and NMR.

Customer Quote:

"We have been extremely impressed by Mitegen's dedication to innovative design and constant attention to the needs of users. The mesh and gripper designs have allowed us to obtain useful data from thin crystals that would otherwise be damaged by mounting on traditional fibre-based loops.

Mitegen's new designs continue to push the envelope for assisting with the consistent and convenient mounting of microcrystals. We look forward to continue working with Mitegen as they continue to refine their designs and respond to the needs of users"

Dr. Ken Ng
Associate Professor
Department of Biological Sciences
University of Calgary

Jena Bioscience product highlight

Phasing Kits - JBS Tungsten Cluster Derivatization Kit

Application
Heavy atom derivatization of biological macromolecules for isomorphous and/or anomalous phasing methods.

Polyoxotungstate clusters have been successfully employed in structural studies because of their high electron-density and very good solubility in aqueous solutions [1]

JBS Phosphotungstate Cluster Kit The kit contains 6 pre-weighed solid aliquots of tri-sodium phosphotungstate clusters at 3.5 mg each. Tri-sodium phosphotungstate contains 12 tungsten metal metal centers bridged by 24 oxygen atoms. In the center of the cluster there is a phosphorous heteroatom surrounded by 4 oxygen atoms, the remaining 12 oxygen atoms forming W=O double bonds.
As a result, the cluster carries a negative charge of 3-.

JBS Metatungstate Cluster Kit The kit contains 6 pre-weighed solid aliquots of hexa-sodium metatungstate clusters at 3.5 mg each. Hexa-sodium metatungstate is composed of 12 tungsten metal centers, each coordinated octahedrally by six oxo O2-ligands. There are a total of 28 bridging oxygen atoms that link the 12 tungsten centers, the remaining 12 oxygen atoms forming W=O double bonds. In the center of the cluster there is a “void” which contains two protons, H+.
As a result, the cluster carries a negative charge of 6-.

JBS Paratungstate Cluster Kit The kit contains 6 pre-weighed solid aliquots of deca-ammonium paratungstate clusters at 3.5 mg each. Deca-ammonium paratungstate is composed of 12 tungsten metal centers, each coordinated octahedrally by six oxo O2-ligands. There are a total of 28 bridging oxygen atoms that link the 12 tungsten centers, the remaining 14 oxygen atoms forming W=O double bonds. In the center of the cluster there is a “void” which contains two protons, H+.
As a result, the cluster carries a negative charge of 10-.

Customer Quote:

"The Mitegen RT system made routine room temperature data collection possible, without the annoyance of capillaries. Now with the mitegen aligner, mounting crystals and the RT tube is easier and more reliable than cryoprotecting!"

James Fraser
qb3@UCSF Fellow
California Institute of Quantitative Biosciences (QB3) and Dept. of Cellular and Molecular Pharmacology
University of California, San Francisco

Recent Press Releases

Spotlight:

Dr. Shane Telfer
Institute of Fundamental Sciences
Massey University
Palmerston North, New Zealand

We are synthetic chemists and our research focuses on metal-organic frameworks (MOFs), which are porous materials constructed from metal ions and bridging organic ligands. A couple of years ago we installed a Rigaku Spider diffractometer that has a 1/4 chi goniometer. Using conventional nylon loop mounts we noticed scattering off the metal pin at certain orientations and were advised to try Mitegen mounts, which place the sample further from the pin, to alleviate this problem. This worked perfectly and we were also impressed by the way the mounts held the sample virtually motionless in the cold stream. The mounts are also useful for estimating the crystal size based on the dimension of the loop.

One of the joys of MOF chemistry is the beauty of the finished product. Here are optical micrographs (right) of some MOF crystals grown from zinc nitrate and functionalized biphenyldicarboxylic acid ligands in DMF under solvothermal conditions. The dimensions of the crystals are several hundred microns. Since they are highly porous, they contain a significant amount of occluded solvent and their transparency is quickly lost if desolvated.

Collecting data on MOF samples is often very challenging due to their tendency to lose crystallinity upon desolvation. Due to the large amount of occluded solvent, in certain cases flash freezing also leads to dreaded diffraction images that resemble my two-year-old daughter's artworks. After many battles with loading and sealing crystals in glass capillaries, interspersed by a few hard-won triumphs, we trialled the MicroRT capillaries. With a steady hand (though a aligner is now available), these polymer sleeves can be loaded with a drop of solvent then slid over a pre-mounted crystal and sealed at the base (we use plasticine). The idea is tremendously simple yet effective and we are now able to screen a bunch of crystals in the same time it took us to coax one into a capillary. Overnight data collection runs are possible with no apparent desolvation or crystal deterioration.

For more information, please see R. K. Deshpande, J. L. Minnaar and S. G. Telfer, Angew. Chem, Int. Ed. 2010, 47, 4598-4602.

Customer Quote:

In my experience, the Microloops LD perform beautifully. Their long, thin-neck design makes is easy to pick up crystals with minimal disturbance to the drop and they are sufficiently robust to use with highly viscous solutions (e.g. cryo-oils) or PEG-skins"

Andrew T. Torelli, Ph.D.MIX Operator
Postdoctoral Associate
Department of Chemtisty abnd Chemical Biology,
Baker Laboratoy, Cornell University

Monthly Tech Tip

Handling and Mounting Needle and Rod Shaped Crystals

Needle and rod shaped crystals pose special challenges. They are easily cracked and broken during retrieval. They are difficult to position over or within the aperture of standard circular-aperture loops. When loop mounted, they tend to be surrounded by too much liquid. This liquid increases background X-ray scatter; it can exert forces that crack the crystal during flash cooling; and it can crystallize to produce ice diffraction rings.

MicroLoops E™ can help you overcome these challenges. These loops have elliptical apertures that more closely match the crystal's shape. Elliptical apertures provide just the right support to minimize cracking during retrieval and mounting, and to minimize excess surrounding liquid. Small flexible fingers within the aperture provide additional support, and allow each aperture to be used with a wider range of crystal sizes.

If cracking still occurs, use a smaller X-ray beam and search for an uncracked region with small mosaicity.

Vertical, horizontal and inclined apertures provide flexibility in sample retrieval and data collection. Vertical apertures tend to be easiest to use. Use a horizontal aperture to gently push on and then slide under a crystal that has adhered to the bottom of a well or to a glass slide

Inclined apertures can provide more efficient data collection. Rod shaped crystals must be carefully oriented in order to collect complete data sets with minimum X-ray exposure. The rod axis often corresponds to a crystal symmetry axis. If this axis lies along the spindle (rotation) axis or if the crystal is triclinic, parts of reciprocal space may be inaccessible. Dauter (Acta Cryst D (1999) 55, 1703-1717) emphasizes the value of collecting complete, high-quality data sets for improved phasing and refinement, and discusses how to orient the symmetry axis relative to the rotation axis for optimal data collection.

MicroLoops E with inclined apertures make it easier to place your crystal near the optimal orientation. To reposition a crystal to a better orientation, try using a MicroTip™ from MicroTools Kit 2. For low-symmetry crystals, use two crystals, one on a vertical loop and one on an inclined loop, to collect a complete data set.

Kappa diffractometers (available on some beamlines) allow the crystal to be rotated about any axis, and provide a more elegant solution to this problem.

Contact Us with comments or suggestions

Customer Quote:

"We have been extremely impressed by Mitegen's dedication to innovative design and constant attention to the needs of users. The mesh and gripper designs have allowed us to obtain useful data from thin crystals that would otherwise be damaged by mounting on traditional fibre-based loops.

Mitegen's new designs continue to push the envelope for assisting with the consistent and convenient mounting of microcrystals. We look forward to continue working with Mitegen as they continue to refine their designs and respond to the needs of users"

Dr. Ken Ng
Associate Professor
Department of Biological Sciences
University of Calgary

Select Recent Citations

Every month, the use on Mitegen products is cited in dozens of papers.

Michael D. Pluth, Maria R. Chan, Lindsey E. McQuade, and Stephen J. Lippard (2011) Seminaphthofluorescein-Based Fluorescent Probes for Imaging Nitric Oxide in Live Cells  Inorganic Chemistry

J. Kmetko, M. Warkentin, U. Englich and R. E. Thorne (2011) Can radiation damage to protein crystals be reduced using small-molecule compounds?  Acta Crystallographica Section D, Biological Crystallography, Volume 67, Part 10 (October 2011)

E. Pellegrini, D. Piano and M. W. Bowler (2011) Direct cryocooling of naked crystals: are cryoprotection agents always necessary?  Acta Crystallographica Section D, Biological Crystallography, Volume 67, Part 10 (October 2011)

Rüdiger W. Seidel, Richard Goddard, Bodo Zibrowius and Iris M. Oppel (2011) A Molecular Antenna Coordination Polymer from Cadmium(II) and 4,4’-Bipyridine Featuring Three Distinct Polymer Strands in the Crysta  Polymers

Cheryl Anne Tanur (2011) Exploring New Synthetic Routes to Frustrated Lewis Pairs  Master's thesis, Dept of Chemistry, Univeristy of Toronto

Joshua L. Allen, Sang-Don Han, Paul D. Boyle, Wesley A. Henderson (2011) Crystal structure and physical properties of lithium difluoro(oxalato)borate (LiDFOB or LiBF2Ox)  Journal of Power Sources

Rajendra P. Singh, Jean’ne M. Shreeve (2011) Synthesis and characterization of novel trifluoromethyl-containing alcohols with Ruppert's reagent  Journal of Fluorine Chemistry

For more citations, click here.

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