<div dir="ltr">Hi Joanna!<div><br></div><div>First, please update to a more recent version of IMOD (4.11.x or 4.12.x). There have been lots of enhancements and bug fixes (including 3D CTF), which will let you get better results. Regarding your other 2 questions, IMOD routinely reconstructs tomograms in this unusual orientation; things get restored to the standard configuration during the final post-processing step, typically by rotating about x (which effectively interchanges the y and z axis but without flipping handedness).</div><div><br></div><div>Best,</div><div>-jh- </div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Mon, Feb 28, 2022 at 1:00 PM <<a href="mailto:3dem-request@ncmir.ucsd.edu">3dem-request@ncmir.ucsd.edu</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">Send 3dem mailing list submissions to<br>
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Today's Topics:<br>
<br>
1. Reconstructing a K3 tomogram (Joanna Brown)<br>
2. Re: Reconstructing a K3 tomogram (Ludtke, Steven J.)<br>
3. Cryo-EM Core Facility Manager Position at Weill Cornell<br>
Medicine (Joel Meyerson)<br>
<br><br><br>---------- Forwarded message ----------<br>From: Joanna Brown <<a href="mailto:joanna.brown@astx.com" target="_blank">joanna.brown@astx.com</a>><br>To: "<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>" <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>Cc: <br>Bcc: <br>Date: Mon, 28 Feb 2022 15:33:41 +0000<br>Subject: [3dem] Reconstructing a K3 tomogram<br>
<div lang="EN-GB" style="overflow-wrap: break-word;">
<div class="gmail-m_-7307828962658267853WordSection1">
<p class="MsoNormal">Hello everyone, <u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">I’m looking for some help reconstructing a tomogram taken on a Krios with a K3 detector.
<u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">I’m doing the reconstruction with imod/etomo v4.9. I’ve taken a relatively low mag tomogram of a 0.6/1 quantifoil hole with particles in it so I can see the particle distribution and ice thickness. I can see the whole foilhole in the field
of view. <u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">I am using patch tracking and everything looks fine with the aligned stacks until I come to the point in the process where a tomogram needs to be computed, including making the sample tomogram to generate the boundary model. (At least I
think it does, but I have to admit, I’m pretty rusty with imod/etomo!)<u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">I notice that the X and the Z axis seem to become switched and I end up with a tomogram of over 1500 Z slices that corresponds to the pixel dimensions of the K3 detector. The reconstruction also becomes very strange, the protein particles
(these look reasonable though strangely arranged relative to each other) visible in a separate plane from the foil hole, which ends up looking like an eye shape instead of a round hole. I tried to compute the tomogram using WBP.
<u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">Thoughts I have had to try to fix problem are:<u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<ol style="margin-top:0cm" start="1" type="1">
<li class="gmail-m_-7307828962658267853MsoListParagraph" style="margin-left:0cm">Try making an initial X,Y boundary model to only include the foil hole for patch tracking and exclude all the carbon and very small particles.
<u></u><u></u></li><li class="gmail-m_-7307828962658267853MsoListParagraph" style="margin-left:0cm">Try a low pass filter?
<u></u><u></u></li><li class="gmail-m_-7307828962658267853MsoListParagraph" style="margin-left:0cm">No idea what to do about the swapping of X and Z dimensions – help!<u></u><u></u></li></ol>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">Any suggestions or pointing out my obvious glaring error/stupidity totally welcome!
<u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">Thanks so much! <u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">Joanna Brown <u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
</div>
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<br><br><br>---------- Forwarded message ----------<br>From: "Ludtke, Steven J." <<a href="mailto:sludtke@bcm.edu" target="_blank">sludtke@bcm.edu</a>><br>To: Joanna Brown <<a href="mailto:joanna.brown@astx.com" target="_blank">joanna.brown@astx.com</a>><br>Cc: "<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>" <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>Bcc: <br>Date: Mon, 28 Feb 2022 15:42:28 +0000<br>Subject: Re: [3dem] Reconstructing a K3 tomogram<br>
<div style="overflow-wrap: break-word;">
Hi Joanna,
<div>while I'm sure there are IMOD solutions to this problem, you might consider trying:</div>
<div><br>
</div>
<div><a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__eman2.org_e2TomoSmall&d=DwMGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=6N7sfHZaBpcerWmtN1_-l1d04aKUt54QeF2j5nE49l3TFxgEzKxiLQEVD_BL4zB2&s=MwUubCDvPDj6bqvz73Cdcv689W8nOTEYvmhTiLGYM48&e=" target="_blank">https://eman2.org/e2TomoSmall</a></div>
<div><br>
</div>
<div>which is fully automated and will produce downsampled tomograms by default, but return to the fully sampled data if you extract local regions (for example for subtomogram averaging). </div>
<div><br>
<div>
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<font face="Courier"><span style="font-size:14px">--------------------------------------------------------------------------------------<br>
Steven Ludtke, Ph.D. <<a href="mailto:sludtke@bcm.edu" target="_blank">sludtke@bcm.edu</a>> Baylor College of Medicine <br>
Charles C. Bell Jr., Professor of Structural Biology<br>
Dept. of Biochemistry and Molecular Biology (<a href="https://urldefense.proofpoint.com/v2/url?u=http-3A__www.bcm.edu_biochem&d=DwMGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=6N7sfHZaBpcerWmtN1_-l1d04aKUt54QeF2j5nE49l3TFxgEzKxiLQEVD_BL4zB2&s=7oxCscH0XQv55BzHQ56DytgpGhs5nGDErj49B3U4T50&e=" target="_blank">www.bcm.edu/biochem</a>)<br>
Academic Director, CryoEM Core (<a href="https://urldefense.proofpoint.com/v2/url?u=http-3A__cryoem.bcm.edu&d=DwMGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=6N7sfHZaBpcerWmtN1_-l1d04aKUt54QeF2j5nE49l3TFxgEzKxiLQEVD_BL4zB2&s=c3N_c2MswvfkmffWYmrECnGnp16qPZ76-SmY2EMzlXw&e=" target="_blank">cryoem.bcm.edu</a>)<br>
Co-Director CIBR Center (<a href="https://urldefense.proofpoint.com/v2/url?u=http-3A__www.bcm.edu_research_cibr&d=DwMGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=6N7sfHZaBpcerWmtN1_-l1d04aKUt54QeF2j5nE49l3TFxgEzKxiLQEVD_BL4zB2&s=iU2lOg-ofxPcbKWVV17FmU6Lit5X3tH-ggvKsVWotII&e=" target="_blank">www.bcm.edu/research/cibr</a>)<br>
<br>
</span></font><br>
</div>
</div>
</div>
</div>
</div>
<div><br>
<blockquote type="cite">
<div>On Feb 28, 2022, at 9:33 AM, Joanna Brown <<a href="mailto:joanna.brown@astx.com" target="_blank">joanna.brown@astx.com</a>> wrote:</div>
<br>
<div><font size="2" style="font-family:Helvetica;font-style:normal;font-variant-caps:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none"><b>***CAUTION:***
This email is not from a BCM Source. Only click links or open attachments you know are safe.</b></font><span style="font-family:Helvetica;font-size:18px;font-style:normal;font-variant-caps:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none;float:none;display:inline"></span>
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<div class="gmail-m_-7307828962658267853WordSection1" style="font-family:Helvetica;font-size:18px;font-style:normal;font-variant-caps:normal;font-weight:normal;letter-spacing:normal;text-align:start;text-indent:0px;text-transform:none;white-space:normal;word-spacing:0px;text-decoration:none">
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
Hello everyone,<span> </span><u></u><u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
I’m looking for some help reconstructing a tomogram taken on a Krios with a K3 detector.<u></u><u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
I’m doing the reconstruction with imod/etomo v4.9. I’ve taken a relatively low mag tomogram of a 0.6/1 quantifoil hole with particles in it so I can see the particle distribution and ice thickness. I can see the whole foilhole in the field of view.<span> </span><u></u><u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
I am using patch tracking and everything looks fine with the aligned stacks until I come to the point in the process where a tomogram needs to be computed, including making the sample tomogram to generate the boundary model. (At least I think it does, but I
have to admit, I’m pretty rusty with imod/etomo!)<u></u><u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
I notice that the X and the Z axis seem to become switched and I end up with a tomogram of over 1500 Z slices that corresponds to the pixel dimensions of the K3 detector. The reconstruction also becomes very strange, the protein particles (these look reasonable
though strangely arranged relative to each other) visible in a separate plane from the foil hole, which ends up looking like an eye shape instead of a round hole. I tried to compute the tomogram using WBP.<u></u><u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
Thoughts I have had to try to fix problem are:<u></u><u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<ol start="1" type="1" style="margin-bottom:0cm;margin-top:0cm">
<li class="gmail-m_-7307828962658267853MsoListParagraph" style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
Try making an initial X,Y boundary model to only include the foil hole for patch tracking and exclude all the carbon and very small particles.<u></u><u></u></li><li class="gmail-m_-7307828962658267853MsoListParagraph" style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
Try a low pass filter?<u></u><u></u></li><li class="gmail-m_-7307828962658267853MsoListParagraph" style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
No idea what to do about the swapping of X and Z dimensions – help!<u></u><u></u></li></ol>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
Any suggestions or pointing out my obvious glaring error/stupidity totally welcome!<u></u><u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
Thanks so much!<span> </span><u></u><u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
Joanna Brown<span> </span><u></u><u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
<div style="margin:0cm;font-size:11pt;font-family:Calibri,sans-serif">
<u></u> <u></u></div>
</div>
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<br><br><br>---------- Forwarded message ----------<br>From: Joel Meyerson <<a href="mailto:jrm2008@med.cornell.edu" target="_blank">jrm2008@med.cornell.edu</a>><br>To: 3dem <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>Cc: <br>Bcc: <br>Date: Mon, 28 Feb 2022 19:59:40 +0000<br>Subject: [3dem] Cryo-EM Core Facility Manager Position at Weill Cornell Medicine<br>
<div lang="EN-US" style="overflow-wrap: break-word;">
<div class="gmail-m_-7307828962658267853WordSection1">
<p class="MsoNormal">Weill Cornell Medicine (WCM) in New York City is hiring a Cryo-EM Core Facility Manager. The position will involve managing the electron microscopy imaging equipment, associated computing infrastructure, and peripherals located in the Cryo-EM
Core Facility.</p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">The facility houses a 200 keV Glacios TEM equipped with a Falcon IV detector and Selectris energy filter and a Vitrobot IV automated EM grid plunge freezer. Data processing is carried out on shared compute nodes managed by the WCM Scientific
Computing Unit.</p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">This position offers opportunities for the incumbent to directly participate in research on the structure and function of macromolecules involved in different biological processes, and interact with other scientists at WCM.</p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">More details about the position and information on how to apply can be found at:</p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal"><a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__career4.successfactors.com_sfcareer_jobreqcareer-3FjobId-3D66908-26company-3DC0000274692P&d=DwQFAg&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=4mBczXjs2xeH0UELegqXtvJuYbxH3UQTuPiIA-d3Jni1_uL8a2OkoVXYyqTVFttL&s=1zh502loIIgenZJEi0oatv7jg3rW23DyuCPbheYQ0sU&e=" target="_blank">https://career4.successfactors.com/sfcareer/jobreqcareer?jobId=66908&company=C0000274692P</a></p>
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