<!DOCTYPE html><html><head><meta http-equiv="Content-Type" content="text/html; charset=utf-8" /></head><body><div data-crea="font-wrapper" style="font-family: Tahoma; font-size: 16px; direction: ltr"><div style="font-family: Tahoma; font-size: 16px"></div>Dear Machinda,<div><br></div><div>are you sure that your particles are intact after freezing? If your protein complex dissociates on the air-water interface, it might look like poor particle spread. Also be aware that text book-like particle spread is desirable, but by no means a necessary requirement to obtain high resolution reconstructions. If you can clearly see your particles, try collecting a small dataset on a better microscope. You might be surprised. </div><div><br></div><div>Other ideas to improve your sample: Crosslinking (this might not be ideal for nucleic acid - protein complexes because the crosslinker can modify lysine residues in the DNA binding interface, so check your crosslinked sample with an EMSA. Crosslinking has the advantage of stabilizing your complex through intermolecular crosslinks and passivating the surface through mono-crosslinks) and detergents (when you use detergents you have to increase the protein concentration, aim for around 2 mg/mL at least).</div><div><br></div><div>Good luck!</div><div><br></div><div>Matthias<br><br><div><div data-crea="font-wrapper" style="font-family: Tahoma; font-size: 16px; direction: ltr">Matthias Vorländer<div>EMBL Heidelberg - Müller Lab<br></div><div>Meyerhofstraße 1, 69117 Heidelberg</div></div></div><br><br><div data-anchor="reply-title">On Mon, Mar 30, 2020 at 14:10, Buerger <buerger@molgen.mpg.de> wrote:</div><blockquote><div><div dir="ltr">
Hi,<br>I would try Quantifoil carbon grids R2/4+ or R3/3+ 2nm additional carbon film. Check allways NS first and then go into cryo. Use 100%humidity and 4C. Blotting only 2 or 4 seconds. If you succeed then you increase the sample concentration by the factor of 10 and freeze 1 or 2sec R1.2/1.3 without carbon. Good luck. I know every grid looks different. Best Jörg.<br><br><div>Am 30. März 2020 05:15:26 MESZ schrieb Satoru Machida <<a href="mailto:dbssator@nus.edu.sg" target="_blank" tabindex="-1" rel="external">dbssator@nus.edu.sg</a>>:<blockquote style="margin: 0pt 0pt 0pt 0.8ex;border-left: 1px solid rgb(204, 204, 204);padding-left: 1ex">
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<span><span>Dear all<br></span></span></div>
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<span><span>I prepared cryo grids for single particle analysis for 600 kD protein-RNA complex. The particles are overcrowded. After dilution, the particles are not separated. When I diluted it further, holes became empty. Gel filtration makes a single peak,
but on the grid they stick together. I already tried glycerol, DMSO, amphipols, salt concentration 70-400 mM. None of them worked well. The buffer contains 150 mM NaCl, 10 mM HEPES pH8, 0.5 mM EDTA, 4 mM DTT. The sample had about 1mg/ml, blotted for 3 sec
, force 0, 100% humidity, 22C on Ultraufoil R0.6/1.0 coated with graphene-oxide. The image was taken by Tecnai12. The particles are hard to get into the holes without graphene-oxide.</span></span></div>
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<span><span>Any expert suggestions will be helpful.</span></span></div>
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<span><span>Kind regards</span></span></div>
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<span><span>Machida</span></span><br></div>
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