<html><head><meta http-equiv="Content-Type" content="text/html charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">Dear Ben<div class="">after reading with a colleague the description (see forwarded message) you sent me, we noticed that the point 2) mentions as “CountTarget” the number of counts per image, while in the script it says #set target counts for tomogram, and the default value is set to 100.</div><div class="">We are wondering:</div><div class="">- is the value referred to the single image or to the whole tomogram?</div><div class="">- is “counts” to be intended as “electrons” or it must be converted into electrons?</div><div class=""><br class=""></div><div class="">We use a K2 summit in super resolution mode, but don’t know which camera was used to test the script (Falcon, CCD, K2 in linear mode?).</div><div class="">Could you give us any clarification?</div><div class="">Best,</div><div class=""><br class=""></div><div class="">Davide</div><div class="">
_________________________________________________<br class=""><br class="">Davide Floris<br class="">PhD student<br class="">Max Planck Institute of Biophysics<br class="">Structural Biology Department<br class="">Max-von-Laue Straße 3<br class="">60438 Frankfurt am Main<br class="">Germany<br class=""><br class="">(L2.090): +49-69-6303-3037<br class="">(B2.205): +49-69-6303-3049
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<br class=""><div><blockquote type="cite" class=""><div class="">On 22 May 2017, at 11:27, Ben Engel <<a href="mailto:bdengel@gmail.com" class="">bdengel@gmail.com</a>> wrote:</div><br class="Apple-interchange-newline"><div class=""><b style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class="">Hi Everyone, </b><span style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""></span><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""><br class=""></div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class="">as promised here is a version of the Hagen tilt scheme that I modified so that it can be started at any starting angle.</div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""><br class=""></div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class="">For lamellae, the pre tilt is 7 º, i.e. if you’re milling at 18º, 11º stage tilt should be your actual ‘0º’. I routinely use a starting angle of 10º,</div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""><br class=""></div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class="">and this results in virtually the same number of counts when tilting +xº and -xº from your actual zero.</div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""><br class=""></div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""><br class=""></div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""><u class="">The procedure to use the script is as follows:</u></div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""><br class=""></div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class="">0) Set up your SerialEM session as usual.</div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""><br class=""></div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class="">1) Copy the contents of the file to an empty macro slot and change parameters as needed. (Once you save your settings the script will be there permanently)</div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""><br class=""></div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class="">2) Check and adjust AngleOffset (your starting angle), CountTarget (the number of counts you’re asking for each image), step (the angle increment) </div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""><br class=""></div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""> and tilttimes (how many times the script will do +step -> -step -> -2*step -> +2*step, e.g. for step = 2, tilt times = 15 you will get ±60º around your actual ‘zero’)</div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""><br class=""></div><div style="font-family: TimesNewRomanPSMT; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; font-size: 12.8px;" class=""> Make sure you’re not exceeding the stage limits!!!</div><br class="Apple-interchange-newline"></div></blockquote></div><br class=""></body></html>