Jan 2010: New crystals

Congratulations to Eoin who, having worked on an NMR structure for the past year on a viral protein that is crucial to replication, has now produced some nice looking crystals of one of its binding partners.

We haven't yet tested them for diffraction but, since Eoin has grown crystals using several different solution conditions, we are pretty hopeful of a successful result.

Jan 2010: New crystals

Congratulations to Adrian, who has been working very hard on his MRes project. He capped it off nicely by producing these beautiful crystals of one of the proteins he was working on. We've just had a first look on the home X-ray source and they diffract to about 3 Å.

Now it's time to hunt for some phases...

Dec 2009: Data collection

Jingjie and Stephen took a quick trip to France to collect data on Jingjie's crystals at the ID23-2 microfocus beamline at the ESRF. A ever, it was a long night's work but we were pleased to come away with a couple of datasets.

Nov 2009: Writing up

Trevor has set down his pipette and picked up his pen to write up his results in his PhD thesis. Or rather, sat down by the keyboard to compose his Magnum Opus.

He seems to be enjoying it... for the time being!

Sept 2009: Melting proteins

Joanna's project had her working up a protocol to investigate the the effects of various mutations on the stability of an important viral protein. We suspect that, unlike many other examples, this protein needs to be relatively unstable to function. To test the impact of each mutation Joanna used differential scanning fluorimetry to look at the melting curves. The results were very interesting - not least because we're not yet sure what they mean!

June 2009: New protease data

Wilfred and Jingjie have been working on new constructs of the FMDV 3C protease to examine different modes of peptide cleavage. Wilfred, using recently reported methods for optimising expression of proteins in E. coli grown in minimal media, has produced some fantastic protein samples. The NMR spectra of a ¹⁵N-labelled sample shown on the right (obtained in collaboration with Steve Matthews' group) shows a clear difference between two 3C^pro constructs that we should be able to investigate structurally.

April 2009: New NMR data

Eoin's crystals, beautiful though they were, appear to be mostly composed of salt. However, by working with Pete Simpson and Rex Kwok in Steve Matthews' NMR group, we have obtained a well-resolved 2D NMR spectrum from a ¹³C,¹⁵N-double-labelled sample of the protein.

The high quality of the spectrum indicates that we should be able to get the solution structure of the protein before too long.

Feb 2009: Outdoor laboratory

On Shrove Tuesday the group decided to conduct some outdoor experiments on the roof of the Blackett Laboratory. These involved an examination of the effects of heat on a mixture of flour, eggs and milk. The results were a bit flat, but tasty nonetheless!

Jan 2009: Working the construct

Eoin has been busy generating a slew of constructs of the viral protein that he is working on. The aim is to produce one that forms a compact, crystallisable domain. Since DNA manipulations are relatively fast and cheap, we can start off by making a series of constructs with different N and C-termini, express them in bacteria and test if the resulting protein expresses well and is soluble.

The gel on the right shows expression tests of three of Eoin's constructs, which differ in the length of the N-terminus (left to right, they start at amino acids 1, 6 and 11). Markers (M) are on the left side of 3 pairs of lanes for uninduced (U) and induced (I) cultures. Clearly all three express well in E. coli.

Update (Feb 09): And within a few days of purification Eoin now has some very nice looking crystals!

Jan 2009: New crystals

Juree is wrapping up her project on expression of viral proteins involved in RNA replication and has managed to round things off with a very nice result. She got enough material to set up a couple of crystallisation trays and obtained some interesting 'hits'. The best crystal (about 0.1 mm long) is shown in the photo on the left.

Update:Our first shot at the crystal (literally) shows that it does indeed diffract. The image below the crystal was taken on the home source and shows diffraction to about 6.7 Å. We need to work on that to get up to closer to 3 Å but it's an excellent start.

Dec 2008: Testing protein expression

Gurdeep has been trying to express a rather tricky viral protein that we think may be associated with membranes. Early tests suggested it was highly toxic to our standard lab strain of E. coli, BL21(DE3).

However, in more recent work, Gurdeep has shown that the protein expresses well in an strain of BL21 that was adapted by John Walker to cope with more toxic proteins, many of them membrane- associated (see paper).

In the western blot presented here, the left-hand pair of lanes shows expression in C43 of a protein of the expected size (~38 kD), while the two lanes to the right show the lack of expression in BL21 cells.

Nov 2008: Probing function

We have been using the structure of the 3C protease from foot-and-mouth disease virus to do mutagenesis experiments to probe the the enzyme in greater detail. Trevor and Jingjie have been generating new mutants to investigate the role of particular amino acids in the active site and key features of the substrate binding site.

The gel on the left shows a cleavage assay. The protein substrate of the reaction is indicated by a green arrow, the 3C enzyme by a blue one and the two major products of the cleavage by red arrows.

July 2008: Purification and cleavage

Dimitra has been helping us to get a new viral enzyme project off the ground. She successfully engineered a number of GST-fusion proteins of the enzyme (since his-tagged constructs had expressed poorly). The proteins now express well in E. coli. The gel on the right shows a first attempt to cleave off the GST tag from the purified proteins using thrombin.

July 2008: More diffraction

Jingjie's crystals (see April, below) didn't get much bigger but they still diffract magnificently. Simon Newstead and David Drew took a few of them along on their recent trip to the micro-focus beamline ID23-2 at the ESRF and came back with a few very nice test images, showing diffraction to about 2Å, even though the crystals are only about 10 µm thick.

July 2008: Grant application

I am in the midst of preparing another grant application - to secure funding for a structural virology project. The key is to prepare a case that persuasively explains what new and fascinating discoveries we hope to make and to lay out the preliminary data that demonstrate the project's feasibility! As you can see, it takes plenty of writing and re-writing...

June 2008: Enzyme assays

Trevor is now working to characterise his viral enzyme using a rather pretty colorimetric assay. The colour changes from yellow to green as the reaction proceeds - the green wells show where the greatest activity lies. The two rows highlighted show the effect of increasing the concentration of an inhibitor.

May 2008: New structure

Well we were right to be optimistic about Amar's crystals since Olga has now collected a 2.7 Å dataset (on the home-source) and phased the data by molecular replacement.

The initial map was rather poor because the inaccurate initial phases (top panel on the right - the map and model don't fit together too well). The refinement started with a free-R value of around 46% but - with much manipulation - Olga has now brought this down to around 30% - producing better maps and a much better model (lower panel)!

Apr 2008: New crystals

Now Jingjie has come up with tantalising crystals of a viral enzyme that she's been working on recently. The photo shows a cluster of slender needles that are only a few µm thick.

Apr 2008: Diffraction!

Olga and Trevor took Amar's crystal (below) to the MPL at Diamond. Using the PX scanner, in which the crystals can be tested for diffraction while they are still in the crystallisation plate, they got a great result (right). The crystals appear nicely ordered and diffract well - note the regularly spaced spots. We're optimistic (for now!) that these crystals will provide us with the data needed to solve the structure.

Feb 2008: New crystals

Amar is currently working on an RNA-binding protein involved in viral translation initiation. He has succeeded in generating a new construct for part of the protein that expresses better than ever before. Better yet, he has managed to grow some crystals which, with a bit of optimization, will hopefully allow us to investigate the structure at high resolution.

Update (Mar 08): Optimization seems to be paying off: the lower photograph shows Amar's latest crystal - much chunkier than the initial hits.

Feb 2008: Mutagenesis results

Trevor is working an interesting viral enzyme. Though the full-length protein is toxic to bacteria, he has been able to express truncated forms that still retain activity. He's now making more mutations, both to increase the chances of getting crystals and to probe the enzyme mechanism. The DNA gel on the right shows that constructs m2-m5 have all been successfully mutated to remove a diagnostic restriction site that is present in the wild-type (wt).

Jan 2008: Expression and purification of a viral enzyme

Much of our work involves efforts to product large amounts (>10 mg) of purified protein. Here, Jingjie has expressed and purified a sample of the 3C protease from FMDV for use in activiy assays.

The gel illustrates the purification of the protein following expression in bacteria. The protease (red arrow) can be separated from the total cell protein (left hand lanes) using a histidine tag.