<html><head><meta http-equiv="Content-Type" content="text/html; charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; line-break: after-white-space;" class="">I am not sure your symmetry expansion will do what you want when starting from a C1 refinement (unless you made sure your reference map is perfectly aligned with the symmetry axis). If you want to do symmetry expansion, you may need a C3 refinement.<div class=""><br class=""></div><div class="">That said, if your C1 refinement can distinguish the two conformations, you probably don’t need symmetry expansion in the first place. Just run 3 classifications with the mask in each protomer position to classify each position. Given your description, this is probably the better approach.</div><div class=""><br class=""></div><div class="">Good luck,<br class=""><div class=""><br class=""></div><div class="">Basil</div><div class=""><br class=""><div><br class=""><blockquote type="cite" class=""><div class="">Am 05.01.2023 um 12:56 schrieb Shashank Khare <<a href="mailto:shashank.khare@u-bordeaux.fr" class="">shashank.khare@u-bordeaux.fr</a>>:</div><br class="Apple-interchange-newline"><div class=""><meta charset="UTF-8" class=""><div style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); 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;" class=""><span lang="EN-GB" style="font-size: 11pt; font-family: Menlo;" class="">Hi,<o:p class=""></o:p></span></div><div style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); 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;" class=""><span lang="EN-GB" style="font-size: 11pt; font-family: Menlo;" class=""><o:p class=""> </o:p></span></div><div style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); 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;" class=""><span lang="EN-GB" style="font-size: 11pt; font-family: Menlo;" class="">I am working on a membrane protein which forms a trimer. Interestingly, the 2 protomers are in outward conformation while the 3<sup class="">rd</sup> protomer is in inward conformation. I am trying to classify the individual protomers using masked 3D classification job without alignment in relion 4 beta. I am using refined particles (C1) as an input for this job.<o:p class=""></o:p></span></div><div style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); 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;" class=""><span lang="EN-GB" style="font-size: 11pt; font-family: Menlo;" class=""><o:p class=""> </o:p></span></div><div style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); 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;" class=""><span lang="EN-GB" style="font-size: 11pt; font-family: Menlo;" class="">There are following pipelines I have tried so far,<o:p class=""></o:p></span></div><div style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); 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;" class=""><span lang="EN-GB" style="font-size: 11pt; font-family: Menlo;" class=""><o:p class=""> </o:p></span></div><div style="margin: 0cm 0cm 0cm 36pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: -18pt;" class=""><span lang="EN-US" style="font-size: 11pt; font-family: Menlo;" class=""><span class="">1.<span style="font-style: normal; font-variant-caps: normal; font-weight: normal; font-stretch: normal; font-size: 7pt; line-height: normal; font-family: "Times New Roman";" class=""> </span></span></span><span lang="EN-US" class="">3D Auto-Refine (C1) </span><span lang="EN-US" style="font-family: Wingdings;" class=""><span class=""></span></span><span lang="EN-US" class=""> Symmetry expansion (C3) </span><span lang="EN-US" style="font-family: Wingdings;" class=""><span class=""></span></span><span lang="EN-US" class=""> Signal subtraction (masking the 3<sup class="">rd</sup> protomer in inward conformation </span><span lang="EN-US" style="font-family: Wingdings;" class=""><span class=""></span></span><span lang="EN-US" class=""> 3D Classification (mask of 3<sup class="">rd</sup> Protomer).<o:p class=""></o:p></span></div><div style="margin: 0cm 0cm 0cm 36pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: -18pt;" class=""><span lang="EN-US" style="font-size: 11pt; font-family: Menlo;" class=""><span class="">2.<span style="font-style: normal; font-variant-caps: normal; font-weight: normal; font-stretch: normal; font-size: 7pt; line-height: normal; font-family: "Times New Roman";" class=""> </span></span></span><span lang="EN-US" class="">3D Auto-Refine(C1) </span><span lang="EN-US" style="font-family: Wingdings;" class=""><span class=""></span></span><span lang="EN-US" class=""> Symmetry expansion (C3) </span><span lang="EN-US" style="font-family: Wingdings;" class=""><span class=""></span></span><span lang="EN-US" class=""> Signal subtraction (invert map created using the 3<sup class="">rd</sup> protomer in inward conformation </span><span lang="EN-US" style="font-family: Wingdings;" class=""><span class=""></span></span><span lang="EN-US" class=""> 3D Classification (mask of 3<sup class="">rd</sup> Protomer).<o:p class=""></o:p></span></div><div style="margin: 0cm 0cm 0cm 36pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: -18pt;" class=""><span lang="EN-US" style="font-size: 11pt; font-family: Menlo;" class=""><span class="">3.<span style="font-style: normal; font-variant-caps: normal; font-weight: normal; font-stretch: normal; font-size: 7pt; line-height: normal; font-family: "Times New Roman";" class=""> </span></span></span><span lang="EN-US" class="">3D Auto-Refine (C1) </span><span lang="EN-US" style="font-family: Wingdings;" class=""><span class=""></span></span><span lang="EN-US" class=""> Symmetry expansion (C3) </span><span lang="EN-US" style="font-family: Wingdings;" class=""><span class=""></span></span><span lang="EN-US" class=""> <span class=""> </span>3D Classification (mask of 3<sup class="">rd</sup> Protomer).<o:p class=""></o:p></span></div><div style="margin: 0cm 0cm 0cm 36pt; font-size: inherit; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); 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;" class=""><span lang="EN-US" class=""><o:p class=""> </o:p></span></div><div style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); 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;" class=""><span lang="EN-US" class="">I have also explored different T values from 10-40 and k values from 4-8. But the output of all of these jobs is only in inward conformation which corresponds to 3<sup class="">rd</sup> protomer.<o:p class=""></o:p></span></div><div style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); 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;" class=""><span lang="EN-US" class=""><o:p class=""> </o:p></span></div><div style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); 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;" class=""><span lang="EN-US" class="">It would be really helpful if someone can guide me with the pipeline here.<o:p class=""></o:p></span></div><div style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); 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;" class=""><span lang="EN-US" class=""><o:p class=""> </o:p></span></div><div style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); 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;" class=""><span lang="EN-US" class="">Regards,<o:p class=""></o:p></span></div><p class="MsoNormal" style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif;"></p><div style="margin: 0cm; font-size: inherit; font-family: Calibri, sans-serif;" class=""><span lang="EN-US" class="">Shashank. <o:p class=""></o:p></span></div>_______________________________________________<br class="">3dem mailing list<br class=""><a href="mailto:3dem@ncmir.ucsd.edu" class="">3dem@ncmir.ucsd.edu</a><br class=""><a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem" class="">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a></div></blockquote></div><br class=""></div></div></body></html>