[ccp4bb] PhD pattern-recognition-for-protein-crystallisation-strategies

Dear CCP4bb, Slightly off topic but perhaps of interest to students. Synopsis: The MARCO (MAchine Recognition of Crystallization Outcomes) project showed that automated image analysis can be used to classify experimental results, having a 94% correct classification rate. Work on a custom

Re: [ccp4bb] challenges in structural biology

Dear CCP4bb, From my industrial perspective: The crystallisation bottleneck has probably been fairly well addressed. High throughput screening using robotics and around 20ul of protein per screen is common. There are lots of screens to try even if they are rather redundant in their content.

Re: [ccp4bb] suggestions for cryoprotectant

Hi Firdous, I had success last week with a condition containing 1.5M MgCl2 which I’d tried cryo-ing with high salt before and got no diffraction. Adding dimethylsulphoxide to a final concentration of 10% v/v in the well solution worked well for me. DMSO was bound in the structure which can be a

Re: [ccp4bb] Precipitation issue during refolding

Maybe this has already been mentioned in which case apologies: I’d try rapid dilution. My only experience of having to refold worked both by dialysis and the dilution method but dilution had the advantage of allowing various conditions to be tested quickly and more controllably at small scale. I

Re: [ccp4bb] Sliconizing of cover-slips

Hi Praveen, 20 years ago I seemed to spend more time siliconising cover slips than setting up the Magic 50. We had special jigs to hold a couple of dozen slips for washing, drying and finally immersion in silane solution. This method was very, very tedious. An alternative method was to pull a

Re: [ccp4bb] Crystals grow at the bottom of the tube.

I have been presented with crystals grown in nmr tubes on two separate occasions. Both diffracted very well and thankfully, neither required a magnetic field for optimisation. On the first occasion I did use the initial sample as a seed stock. Best wishes, David From: CCP4 bulletin board

Re: [ccp4bb] DNA RNA annealing problem

Hi Weifei, When I was looking at Holliday junction annealing I found that the salt concentration was important. NaCl around 300-500mM improved yield as did MgCl2 at lower (100-200mM) concentrations. So, it might be worth trying higher salt. Dave -Original Message- From: CCP4 bulletin

Re: [ccp4bb] Tev cleavage

You could try adding TEV to your protein just prior to crystallisation. I produced 2 equivalent structures this way; one with the tag clearly visible packing between molecules in an I centred space group and a 2nd (with TEV added) in P21 where the tag was no longer visible (the packing

Re: [ccp4bb] Thin plate crystals

The crystals don't look that bad in my opinion. Maybe the cryo step is sub optimal? Try using large loops (reduces surface tension forces during transfer). Butane 2,3 diol is a good cryo to try. Maybe shoot them at room temp (in situ) to get an idea of how they diffract before you manipulate

Re: [ccp4bb] Crystallization issue

On the face of it, I wouldn’t get overly excited by the drop. However, I’ve been underwhelmed and wrong before. If you’ve got access to a synchrotron beamline that can shoot the material in-situ it might be a good experiment to try. Best of luck, Dave Dr. David Hargreaves Associate Principal

Re: [ccp4bb] Solvent channels

Hi Reza, I can recommend the work of Marc-Olivier Coppens and Kourosh Malek and the references therein for an interesting journey through solvent channels. David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery

Re: [ccp4bb] Invisible atoms in ligands

Hi All, I had an interesting case recently where a Cl atom of a chlorophenyl moiety went missing in a structure (primary X-ray damage was the suspected culprit). Small molecule Mass Spec suggested the atom was there to start with but it was quite obviously missing in the maps (1.9Ang) and the

[ccp4bb] Kabat, insertion codes refinement

Dear CCP4bb, I'm refining an antibody structure which requires Kabat residue numbering with insertion codes. My setup of Refmac5 and Buster both break peptide bonds between some (not all) of the residues with insertion codes. I was wondering whether there is a special way of handling these

[ccp4bb] pdb insertion codes

Dear CCP4bb, Does anyone know a simple way to get rid of residue number insertion codes and then renumber residues normally in pdb files? Help much appreciated, David David Hargreaves Associate Principal Scientist _

[ccp4bb] Crymon

Dear CCP4bb, Does anyone know who (if anyone) supports Crystal Miner (Emerald / Decode). Kind Regards, David David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics Mereside,

[ccp4bb] Calculated vs Measured pI

Dear CCP4bbers, I was wondering whether anyone knew of a program that would calculate the pI of a protein based on its structure rather than its sequence and whether such a calculation might correlate better with the measured pI Dave David Hargreaves Associate Principal Scientist

Re: [ccp4bb] salt or not?

Dear Careina, I would be cautious of using dyes. Much better to 1) try in-situ diffraction if possible as this is least invasive or 2) pick a sensible cryo and just freeze the crystal(s). I would try to same something from the experiment for seed stock possibly sequencing. Dave David

Re: [ccp4bb] crystal dehydration

Judith, If you can, try monitoring the diffraction during the dehydration experiment. There are plenty of ways of doing this. Perhaps the simplest is to shoot your crystals in-situ after swapping the precipitant for a more concentrated one. The is also the Free Mounting System. David

Re: [ccp4bb] To cryo or not to cryo...

Hi Yuri, I've had a very similar experience last week which ended badly. I opted to cryo my new crystals using my favourite cryo. Diffraction showed at least the crystals were protein but not much more. A classic example of cryo killing crystals. I mounted the remaining tiny fragment from the

Re: [ccp4bb] Problems in crystallization

I've seen this effect too. In the case I saw the drops were growing (and spreading) due to a reverse diffusion gradient. Optimised conditions used salting in method i.e. crystallising as the [pptnt] decreases during equilibration due to dilution as water is Tx from well to drop. Rare but not

Re: [ccp4bb] Question about weird diffraction map

Hi Zhao, I would try a crystal that has not been treated with any dye. Small molecules, particularly drug like compounds, crystallise out in soaking experiments and can give diffraction similar to yours. My worry is that you have non-diffracting protein crystals coated in some small molecule

Re: [ccp4bb] The effect of His-tag location on crystallization

In trying to reproduce a very nice public structure a cloning mis-hap put a -GS- prior to the C-term 6His tag. The resultant crystals had a 500Ang C dimension and 16 molecules in the au. Even a single amino acid could make all the difference David Hargreaves Associate Principal Scientist

Re: [ccp4bb] Crystallization with antibody

There are plenty of reports (some even successful) where small concentrations of proteases have been used to enable crystallisation (with soluble proteins). Having put the effort into getting your material in the first place it's a small extra step to try. The professional way is to run a time

Re: [ccp4bb] effect of Izit dye within the crystal structure

I've collected data from crystal soaked with brightly coloured ligands which partition into the solvent channels. Although the crystal becomes highly coloured (mopping up most of the colour from the drop) the structures remained resolutely empty. I am intrigued by this observation and wonder how

Re: [ccp4bb] Protein preps become a jelly

Hi Aidong, I've seen this in protein samples that have been snap frozen then thawed on ice. Thawing at room temperature (or in the hand) stopped this state forming. Could temperature be the issue? Maybe try 10C or leave the samples on the bench for a while and see if the state is reversible?

Re: [ccp4bb] Aging PEGs

I went through the peg solid stocks in our cupboard and noticed some of them smelled acrid. I probably won’t use them. Not sure whether they’re chemically decomposing. Maybe fossilising? Dave David Hargreaves Associate Principal Scientist

Re: [ccp4bb] Methods for dehydrating crystals

Hi Andrea, If you haven't already done so it might be worth trying a room temperature mount (capillary or in-situ) to get a feeling for how well the crystals diffract to start with. Dave David Hargreaves Associate Principal Scientist

Re: [ccp4bb] Bypassing phase separation for nice crystals.

Hi Timur, I've had a crystal which sounds similar to yours. When I came to freeze it noticed that it was not exactly solid more amorphous like treacle. Laughingly I went ahead and screened it anyway and was shocked to see diffraction to around 15Ang. I was lucky enough to have other crystals in

Re: [ccp4bb] generate large symmetry model

), followed by a second call using symop/symcommit to apply unit cell translations. Cheers Martyn From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hargreaves, David Sent: 30 June 2011 13:52 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] generate large symmetry model Does anyone

[ccp4bb] generate large symmetry model

Does anyone have a rigorous method (or script) for generating an extended lattice e.g 3x3x3 unit cells from any pdb file? Any help gratefully received, Dave David Hargreaves Associate Principal Scientist _ AstraZeneca

Re: [ccp4bb] Preparation of seed-stocks without seed-beads

Take two siliconised glass cover slips. Place 2-5ul of mother liquor on one and transfer a single crystal to this solution. Place the other cover slip on top and press down. The two optically flat surfaces crush the crystal (best observed using a microscope). Slide the two cover slips apart and

[ccp4bb] Temporary Postdoctoral Research Scientist at AstraZeneca, Alderley Park

Dear CCP4bb, I like to take this opportunity to draw your attention to the following role (below). Please follow the link to the AstraZeneca web site (job search DECS156) for more details and application forms. (please don't apply via this email!) David Hargreaves