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</style><p class="MsoNormal" style="margin: 0cm; font-size: medium; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); color: rgb(0, 0, 0); font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;"><span lang="EN-GB" style="font-size: 11pt; font-family: Menlo; color: black;">Hi,<o:p></o:p></span></p><p class="MsoNormal" style="margin: 0cm; font-size: medium; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); color: rgb(0, 0, 0); font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;"><span lang="EN-GB" style="font-size: 11pt; font-family: Menlo; color: black;"><o:p> </o:p></span></p><p class="MsoNormal" style="margin: 0cm; font-size: medium; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); color: rgb(0, 0, 0); font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;"><span lang="EN-GB" style="font-size: 11pt; font-family: Menlo; color: black;">I am working on a membrane protein which forms a trimer. Interestingly, the 2 protomers are in outward conformation while the 3<sup>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></o:p></span></p><p class="MsoNormal" style="margin: 0cm; font-size: medium; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); color: rgb(0, 0, 0); font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;"><span lang="EN-GB" style="font-size: 11pt; font-family: Menlo; color: black;"><o:p> </o:p></span></p><p class="MsoNormal" style="margin: 0cm; font-size: medium; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); color: rgb(0, 0, 0); font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;"><span lang="EN-GB" style="font-size: 11pt; font-family: Menlo; color: black;">There are following pipelines I have tried so far,<o:p></o:p></span></p><p class="MsoNormal" style="margin: 0cm; font-size: medium; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); color: rgb(0, 0, 0); font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;"><span lang="EN-GB" style="font-size: 11pt; font-family: Menlo; color: black;"><o:p> </o:p></span></p><p class="MsoListParagraphCxSpFirst" style="text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-size:11.0pt;mso-bidi-font-size:12.0pt;font-family:Menlo;
mso-fareast-font-family:Menlo;color:black"><span style="mso-list:Ignore">1.<span style="font:7.0pt "Times New Roman""> </span></span></span><!--[endif]--><span lang="EN-US">3D Auto-Refine (C1) </span><span lang="EN-US" style="font-family: Wingdings;"><span></span></span><span lang="EN-US"> Symmetry expansion (C3) </span><span lang="EN-US" style="font-family: Wingdings;"><span></span></span><span lang="EN-US"> Signal subtraction (masking the 3<sup>rd</sup> protomer in inward conformation </span><span lang="EN-US" style="font-family: Wingdings;"><span></span></span><span lang="EN-US"> 3D Classification (mask of 3<sup>rd</sup> Protomer).<o:p></o:p></span></p><p class="MsoListParagraphCxSpMiddle" style="text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-size:11.0pt;mso-bidi-font-size:12.0pt;font-family:Menlo;
mso-fareast-font-family:Menlo;color:black"><span style="mso-list:Ignore">2.<span style="font:7.0pt "Times New Roman""> </span></span></span><!--[endif]--><span lang="EN-US">3D Auto-Refine(C1) </span><span lang="EN-US" style="font-family: Wingdings;"><span></span></span><span lang="EN-US"> Symmetry expansion (C3) </span><span lang="EN-US" style="font-family: Wingdings;"><span></span></span><span lang="EN-US"> Signal subtraction (invert map created using the 3<sup>rd</sup> protomer in inward conformation </span><span lang="EN-US" style="font-family: Wingdings;"><span></span></span><span lang="EN-US"> 3D Classification (mask of 3<sup>rd</sup> Protomer).<o:p></o:p></span></p><p class="MsoListParagraphCxSpMiddle" style="text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-size:11.0pt;mso-bidi-font-size:12.0pt;font-family:Menlo;
mso-fareast-font-family:Menlo;color:black"><span style="mso-list:Ignore">3.<span style="font:7.0pt "Times New Roman""> </span></span></span><!--[endif]--><span lang="EN-US">3D Auto-Refine (C1) </span><span lang="EN-US" style="font-family: Wingdings;"><span></span></span><span lang="EN-US"> Symmetry expansion (C3) </span><span lang="EN-US" style="font-family: Wingdings;"><span></span></span><span lang="EN-US"> <span> </span>3D Classification (mask of 3<sup>rd</sup> Protomer).<o:p></o:p></span></p><p class="MsoListParagraphCxSpLast" style="margin: 0cm 0cm 0cm 36pt; font-size: medium; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); color: rgb(0, 0, 0); font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;"><span lang="EN-US"><o:p> </o:p></span></p><p class="MsoNormal" style="margin: 0cm; font-size: medium; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); color: rgb(0, 0, 0); font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;"><span lang="EN-US">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>rd</sup> protomer.<o:p></o:p></span></p><p class="MsoNormal" style="margin: 0cm; font-size: medium; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); color: rgb(0, 0, 0); font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;"><span lang="EN-US"><o:p> </o:p></span></p><p class="MsoNormal" style="margin: 0cm; font-size: medium; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); color: rgb(0, 0, 0); font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;"><span lang="EN-US">It would be really helpful if someone can guide me with the pipeline here.<o:p></o:p></span></p><p class="MsoNormal" style="margin: 0cm; font-size: medium; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); color: rgb(0, 0, 0); font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;"><span lang="EN-US"><o:p> </o:p></span></p><p class="MsoNormal" style="margin: 0cm; font-size: medium; font-family: Calibri, sans-serif; caret-color: rgb(0, 0, 0); color: rgb(0, 0, 0); font-style: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; text-decoration: none;"><span lang="EN-US">Regards,<o:p></o:p></span></p><p class="MsoNormal" style="font-size: medium;"><style class="WebKit-mso-list-quirks-style">
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text-indent:-9.0pt;}
@list l0:level7
{mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-18.0pt;}
@list l0:level8
{mso-level-number-format:alpha-lower;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-18.0pt;}
@list l0:level9
{mso-level-number-format:roman-lower;
mso-level-tab-stop:none;
mso-level-number-position:right;
text-indent:-9.0pt;}
-->
</style></p><p class="MsoNormal" style="font-size: medium;"><span lang="EN-US">Shashank. <o:p></o:p></span></p></body></html>