<html><head><meta http-equiv="content-type" content="text/html; charset=utf-8"></head><body style="overflow-wrap: break-word; -webkit-nbsp-mode: space; line-break: after-white-space;"><div></div><div><br></div><div><br></div><div><br><blockquote type="cite"><div>On Apr 13, 2023, at 7:36 AM, Mariena Silvestry Ramos <ms3289@cornell.edu> wrote:</div><br class="Apple-interchange-newline"><div><meta charset="UTF-8"><span style="caret-color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none; float: none; display: inline !important;">Hi James and all,</span><div style="caret-color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;"><br></div><div style="caret-color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;">I agree. I've seen "webby" stuff like that too, even in protein preps, which I've attributed in the past to denatured/mesbehaving protein. Unfortunately I don't have an x-ray detector on any of my columns to quickly check what's in them, but that would be a possibility in a system that does have it. </div><div style="caret-color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;"><br></div><div style="caret-color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;">The items in question do look like micelles, and as Vincent pointed out, we don't have info on what the contents/combo of lipids and detergents are. </div><div style="caret-color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;"><br></div><div style="caret-color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;">Best,</div><div style="caret-color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;"><br></div><div style="caret-color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration: none;">Mariena<br><br><div dir="ltr">Sent from my Game Boy</div><div dir="ltr"><br><blockquote type="cite">On Apr 13, 2023, at 10:08 AM, David Michael Belnap <David.Belnap@utah.edu> wrote:<br><br></blockquote></div><blockquote type="cite"><div dir="ltr"><div class="WordSection1" style="page: WordSection1;"><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif;"><span style="font-family: Georgia, serif;">James,<o:p></o:p></span></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif;"><span style="font-family: Georgia, serif;"><o:p> </o:p></span></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif;"><span style="font-family: Georgia, serif;">Those images look consistent with large particles that exclude water. I don’t know the expected sizes of micelles, however. If the appearance changes with increased exposure to the electron beam, then I would agree that you have beam damage. Be careful of claims of “pure” samples. I often see much more than what is claimed to be in a pure sample. Is what you are looking for supposed to be smaller than these particles? As Vincent suggests, you may have constructs different from what you expect. Your lower magnification picture suggests a diluted sample to me.<o:p></o:p></span></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif;"><span style="font-family: Georgia, serif;"><o:p> </o:p></span></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif;"><span style="font-family: Georgia, serif;">David<o:p></o:p></span></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif;"><span style="font-family: Georgia, serif;"><o:p> </o:p></span></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif;"><span style="font-family: Georgia, serif;"><o:p> </o:p></span></div><div style="border-style: solid none none; border-top-width: 1pt; border-top-color: rgb(181, 196, 223); padding: 3pt 0in 0in;"><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif;"><b><span style="font-size: 12pt;">From:<span class="Apple-converted-space"> </span></span></b><span style="font-size: 12pt;">3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of vincent Chaptal <vincent.chaptal@ibcp.fr><br><b>Date:<span class="Apple-converted-space"> </span></b>Thursday, April 13, 2023 at 7:58 AM<br><b>To:<span class="Apple-converted-space"> </span></b>"Kizziah, James" <kizziah4@uab.edu>, "3dem@ncmir.ucsd.edu" <3dem@ncmir.ucsd.edu><br><b>Subject:<span class="Apple-converted-space"> </span></b>Re: [3dem] Interpretation of strange objects in a cryo-EM sample<o:p></o:p></span></div></div><div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif;"><o:p> </o:p></div></div><p class="MsoNormal" style="margin: 0in 0in 12pt; font-size: 11pt; font-family: Calibri, sans-serif;"><span style="font-size: 13.5pt; font-family: "Courier New";">Dear James,<span class="Apple-converted-space"> </span><br><br>you didn't say which detergent the micelles would be orinigating from, it changes from detergent to detergent.<span class="Apple-converted-space"> </span><br><br>But in any case, beside the mix DDM/CHS which forms small micelles when concentrated but large objects when diluted, most micelles are much much smaller, say 30-50Å or so.<span class="Apple-converted-space"> </span><br><br>I think there is an article by the group of Arne Moeller on detergent micelle observation by negative stain, I don't recall if they tried in cryo.<span class="Apple-converted-space"> </span><br><br>Best<br>Vincent</span><o:p></o:p></p><div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif;">Le 13/04/2023 à 15:48, Kizziah, James a écrit :<o:p></o:p></div></div><blockquote style="margin-top: 5pt; margin-bottom: 5pt;"><div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif; line-height: 17.6px;">Hi all,<o:p></o:p></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif; line-height: 17.6px;"> <o:p></o:p></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif; line-height: 17.6px;">We have imaged a cryo-EM sample presumed to contain micelles and observed the large, white objects full of/covered with junk shown in the attached images. Our best guess is that these are (or were) the micelles and have been destroyed by the electron beam, contact with the air water interface, or both, after vitrification. Has anyone seen something like this before or does anyone have alternative interpretations?<o:p></o:p></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif; line-height: 17.6px;"> <o:p></o:p></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif; line-height: 17.6px;">Glow-discharged lacey carbon grids plunge frozen with a Mark IV Vitrobot, clipped and imaged on a Glacios.<o:p></o:p></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif; line-height: 17.6px;"> <o:p></o:p></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif; line-height: 17.6px;">Thanks,<o:p></o:p></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif; line-height: 17.6px;">James<o:p></o:p></div></div><div style="margin: 0in; font-size: 11pt; font-family: Calibri, sans-serif;"><br><br><o:p></o:p></div><pre style="margin: 0in 0in 0.0001pt; font-size: 10pt; font-family: "Courier New";">_______________________________________________<o:p></o:p></pre><pre style="margin: 0in 0in 0.0001pt; 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