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Dear JL,

Years ago, this was a common problem when I was crystallizing myosin

constructs for my doctoral work. Some of the most beautiful crystals I

got showed little or no diffraction. Often this occurs when there is a very

large water content in the asymmetric unit and in proteins that have a

great deal of intrinsic disorder. Jon's suggestion to flash-cool them in

oil could work. Your high PEG concentration in the flash-cooling solution

might work, too, but you probably cannot just plunge your crystals

suddenly into a much higher PEG solution. A few suggestions:

1) Whichever cryoprotectant you use, introduce it to your crystal GRADUALLY,

such as in steps (e.g., 10% ==> 15% ==> 20% ==>25%) or something like that.

For some proteins, drastic rapid changes of concentrations of anything in

the solution can damage the crystal and introduce enough disorder so that

the protein crystal will not diffract well. Sometimes you can tell when there's

damage if the crystal cracks (or melts away), but not always.

2) You may have to experiment with different cryoprotectants. Different

cryoprotectants make different protein crystals happy. For example, try PEG 200,

PEG 400, PEG 600, glycerol, sucrose, trehalose, other disaccharide sugars, MPD, butanediols.

3) I have found that, as a "rule of thumb", 25% of any cryoprotectant is enough

to protect against ice formation. If your protein crystal can tolerate it, higher

concentrations could be better (because they can shrink the unit cell and reduce

some of the water content). HOWEVER, many protein crystals will not tolerate

much higher concentrations without taking on damage.

I hope this helps.

-Daniel

On Tue, Aug 14, 2018 at 9:45 AM, ferrer <[log in to unmask]> wrote:

Hi

Did you try them at room temp, in situ (straight in the plate). We observe that time to time on our beamline, when just harvesting, not mentioning cryo protection, is enough to loose all diffraction. It-s rare but happens.

Regards

JL

On 14/08/2018 11:58, Careina Edgooms wrote:

I got the most beautiful crystals I have ever seen and they don't diffract at all. Not poor diffraction, NO diffraction. Anyone know why this could be and how I can go about fixing it? I had three beautiful crystals and not one diffracted. I did leave them in the drop for about 3 weeks before harvesting and in liquid nitrogen for about a month before diffracting. Could that be a factor? If I regrew more beautiful crystals and diffracted straight away could that help?

Careina

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Jean-Luc Ferrer                 
Institut de Biologie Structurale
71 Avenue des Martyrs           
CS 10090                        
38044 Grenoble Cedex 9 - FRANCE

Ph.:  +33 (0)4 57 42 85 22
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