<div dir="ltr"><div dir="ltr">Hi tomo users,</div><div>If you used different magnifications for the data collection, the readout of the tilt angle would be different. Check your .mdoc files at LMM, MMM, and high mag for the tilt series, you may get some clues.</div><div>Best,</div><div>Shiwei</div><div><br></div><div dir="ltr"><br clear="all"><div><div dir="ltr" class="gmail_signature"><div dir="ltr"><div>Shiwei Zhu, Ph.D.</div>Assistant Professor, <div>Department of Physiology and Biophysics<div>Case Western Reserve University</div><div>School of Medicine, Robbins Building E621</div><div>Email: <a href="mailto:shiwei.zhu@case.edu" target="_blank">shiwei.zhu@case.edu</a></div><div><a href="https://urldefense.com/v3/__https://www.shiweizhulab.org__;!!Mih3wA!CCkeh8VvKrH1qQN9f1QdCF2-bFDMWGWWsYZZVls_JUdYznwGc85zIZVdIfhOZBnwpmXcUGyhAtdiI8ZynA$" target="_blank">https://www.shiweizhulab.org</a><br></div><div><a href="https://urldefense.com/v3/__https://physiology.case.edu/people/faculty/shiwei-zhu__;!!Mih3wA!CCkeh8VvKrH1qQN9f1QdCF2-bFDMWGWWsYZZVls_JUdYznwGc85zIZVdIfhOZBnwpmXcUGyhAte8gn3wxQ$" target="_blank">https://physiology.case.edu/people/faculty/shiwei-zhu</a><br></div><div><br></div><div><br></div></div><div><br></div></div></div></div><br></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Mon, Mar 18, 2024 at 3:00 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>
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1. Re: Tilt axis angle (Sylvain Trepout)<br>
2. Re: Tilt axis angle (Ricardo Righetto)<br>
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Message: 1<br>
Date: Mon, 18 Mar 2024 12:57:32 +1100<br>
From: Sylvain Trepout <<a href="mailto:sylvain.trepout@monash.edu" target="_blank">sylvain.trepout@monash.edu</a>><br>
To: Zongli Li <<a href="mailto:zongli.li@gmail.com" target="_blank">zongli.li@gmail.com</a>><br>
Cc: 3dem <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
Subject: Re: [3dem] Tilt axis angle<br>
Message-ID:<br>
<<a href="mailto:CAGOVMwH4uZFUqj9gVJ_jCR42e4ZngGVcXo5sZCrm969sLjTGCg@mail.gmail.com" target="_blank">CAGOVMwH4uZFUqj9gVJ_jCR42e4ZngGVcXo5sZCrm969sLjTGCg@mail.gmail.com</a>><br>
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<br>
Hi Zongli and Ricardo, and tomo enthusiasts,<br>
<br>
Same here, on a Krios G4 with Falcon 4i and Tomo software version<br>
5.16.0.6389, the orientation of the tilt axis is not reported correctly.<br>
TiltAxisAngle = -174.72<br>
RotationAngle = 84.72<br>
<br>
But the correct tilt axis angle found by IMOD is 95.07<br>
It does not fit the formula you gave Ricardo though... off by just 10<br>
degrees.<br>
Or maybe this is a flip and then the orientation would be ok, but this<br>
would change the handedness.<br>
<br>
For comparison, on the Krios G1 (with a K3 and an older version of Tomo<br>
software)<br>
TiltAxisAngle = -174.47<br>
RotationAngle = -84.72<br>
The correct tilt axis orientation (as found by IMOD) is around 87 (this<br>
fits your formula, Ricardo).<br>
<br>
Best wishes,<br>
Sylvain<br>
<br>
PhD<br>
Research Fellow - Microscopy Australia<br>
<br>
*Monash University*<br>
Ramaciotti Centre for Cryo-EM<br>
G92, 15 Innovation Walk, Clayton Campus<br>
Victoria, 3800 Australia<br>
<br>
E: <a href="mailto:sylvain.trepout@monash.edu" target="_blank">sylvain.trepout@monash.edu</a><br>
<br>
<br>
On Sun, 17 Mar 2024 at 07:53, Zongli Li <<a href="mailto:zongli.li@gmail.com" target="_blank">zongli.li@gmail.com</a>> wrote:<br>
<br>
> Dear Ricardo,<br>
><br>
> Thank you so much! This makes a lot of sense.<br>
> Best wishes,<br>
><br>
> Zongli<br>
><br>
> On Sat, Mar 16, 2024 at 12:37?PM Ricardo Righetto <<br>
> <a href="mailto:ricardorighetto@gmail.com" target="_blank">ricardorighetto@gmail.com</a>> wrote:<br>
><br>
>> Dear Zongli,<br>
>><br>
>> Thanks for bringing this up. As far as I know this is a bug (?) in Tomo5.<br>
>> This is how you convert the TiltAxisAngle from the Tomo5 mdoc to match the<br>
>> IMOD/SerialEM convention (followed by RELION and other packages):<br>
>><br>
>> TiltAxisAngle_SerialEM = TiltAxisAngle_Tomo5 * (-1) - 90<br>
>><br>
>> Thanks to Grigory Sharov for reporting this conversion previously on the<br>
>> EMAN2 mailing list:<br>
>> <a href="https://urldefense.com/v3/__https://groups.google.com/g/eman2/c/piux0GdX_7M/m/ScQNh9TNAQAJ__;!!Mih3wA!Hpk_Da05ULp8VSDgCGDsUn4lxlUYtLSBQcdl-AumgluDZ0DfzRUu0vBVT4ETXJOa9ZjzKhYlIBKictUVVxFob3BkJ-5voQgo$" rel="noreferrer" target="_blank">https://urldefense.com/v3/__https://groups.google.com/g/eman2/c/piux0GdX_7M/m/ScQNh9TNAQAJ__;!!Mih3wA!Hpk_Da05ULp8VSDgCGDsUn4lxlUYtLSBQcdl-AumgluDZ0DfzRUu0vBVT4ETXJOa9ZjzKhYlIBKictUVVxFob3BkJ-5voQgo$</a> <br>
>> <<a href="https://urldefense.com/v3/__https://groups.google.com/g/eman2/c/piux0GdX_7M/m/ScQNh9TNAQAJ__;!!Mih3wA!Go-c8e-QwJVBsU0qttyzgSDv-knvzqN6KJbTdN11SKI-RKgQ9dktWrwtqL_J9HC10IqJx5KxDD2MNvPZcrYB$" rel="noreferrer" target="_blank">https://urldefense.com/v3/__https://groups.google.com/g/eman2/c/piux0GdX_7M/m/ScQNh9TNAQAJ__;!!Mih3wA!Go-c8e-QwJVBsU0qttyzgSDv-knvzqN6KJbTdN11SKI-RKgQ9dktWrwtqL_J9HC10IqJx5KxDD2MNvPZcrYB$</a>><br>
>><br>
>> TiltAxisAngle and RotationAngle should always be 90 degrees apart in any<br>
>> mdoc:<br>
>> <a href="https://urldefense.com/v3/__https://bio3d.colorado.edu/SerialEM/hlp/html/about_formats.htm__;!!Mih3wA!Hpk_Da05ULp8VSDgCGDsUn4lxlUYtLSBQcdl-AumgluDZ0DfzRUu0vBVT4ETXJOa9ZjzKhYlIBKictUVVxFob3BkJ6545NyN$" rel="noreferrer" target="_blank">https://urldefense.com/v3/__https://bio3d.colorado.edu/SerialEM/hlp/html/about_formats.htm__;!!Mih3wA!Hpk_Da05ULp8VSDgCGDsUn4lxlUYtLSBQcdl-AumgluDZ0DfzRUu0vBVT4ETXJOa9ZjzKhYlIBKictUVVxFob3BkJ6545NyN$</a> <br>
>> <<a href="https://urldefense.com/v3/__https://bio3d.colorado.edu/SerialEM/hlp/html/about_formats.htm__;!!Mih3wA!Go-c8e-QwJVBsU0qttyzgSDv-knvzqN6KJbTdN11SKI-RKgQ9dktWrwtqL_J9HC10IqJx5KxDD2MNiJtThea$" rel="noreferrer" target="_blank">https://urldefense.com/v3/__https://bio3d.colorado.edu/SerialEM/hlp/html/about_formats.htm__;!!Mih3wA!Go-c8e-QwJVBsU0qttyzgSDv-knvzqN6KJbTdN11SKI-RKgQ9dktWrwtqL_J9HC10IqJx5KxDD2MNiJtThea$</a>><br>
>> That's why you got the "correct" result when using the value from<br>
>> RotationAngle as the tilt axis. Beware though that you may have inverted<br>
>> handedness in your reconstructions. In my experience, the conversion above<br>
>> should give the correct results.<br>
>><br>
>> Would be great if others can comment if this is indeed the case, and why<br>
>> this bug persists for so long...<br>
>><br>
>> Best wishes,<br>
>><br>
>> --<br>
>> Ricardo Diogo Righetto<br>
>><br>
>><br>
>> Em sex., 15 de mar. de 2024 ?s 21:33, Zongli Li <<a href="mailto:zongli.li@gmail.com" target="_blank">zongli.li@gmail.com</a>><br>
>> escreveu:<br>
>><br>
>>> Dear colleagues,<br>
>>><br>
>>> We recently collected some tilt series data on vitrified ribosome sample<br>
>>> using ThermoFisher Scientifc's Tomo5 software. The data were collected on<br>
>>> Falcon 4i. When trying to process the data using the new version of relion<br>
>>> tomo workflow (relion 5.0-beta), we are wondering what parameter we should<br>
>>> use from the dmoc file for tilt axis angle (TiltAxisAngle or<br>
>>> RotationAngle). If the TiltAxisAngle is used in our test run, the<br>
>>> reconstructed tomogram is nonsense, but the reconstructed tomogram looks<br>
>>> okay if RotationAngle is used. Any suggestions or explanations on this<br>
>>> would be highly appreciated.<br>
>>><br>
>>> Best,<br>
>>><br>
>>> Zongli<br>
>>> _______________________________________________<br>
>>> 3dem mailing list<br>
>>> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a><br>
>>> <a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem" rel="noreferrer" target="_blank">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a><br>
>>><br>
>> _______________________________________________<br>
> 3dem mailing list<br>
> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a><br>
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<br>
Message: 2<br>
Date: Mon, 18 Mar 2024 09:46:47 +0100<br>
From: Ricardo Righetto <<a href="mailto:ricardorighetto@gmail.com" target="_blank">ricardorighetto@gmail.com</a>><br>
To: Sylvain Trepout <<a href="mailto:sylvain.trepout@monash.edu" target="_blank">sylvain.trepout@monash.edu</a>><br>
Cc: 3dem <<a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a>><br>
Subject: Re: [3dem] Tilt axis angle<br>
Message-ID:<br>
<<a href="mailto:CAB7-RrtgOZnd1nzJTEMyN4NKvd6txkZ2oqv3EawBFU9iR9Hc5A@mail.gmail.com" target="_blank">CAB7-RrtgOZnd1nzJTEMyN4NKvd6txkZ2oqv3EawBFU9iR9Hc5A@mail.gmail.com</a>><br>
Content-Type: text/plain; charset="utf-8"<br>
<br>
Hi Sylvain,<br>
<br>
> Or maybe this is a flip and then the orientation would be ok, but this<br>
would change the handedness.<br>
Yes, this is what I think might be happening here. This "off by just 10<br>
degrees" is misleading, it's actually the expected 5 degrees deviation from<br>
vertical, but going over to the other side of the axis. Your tilt series<br>
images are probably being flipped somewhere in your pipeline (perhaps at<br>
the detector itself).<br>
Which is not necessarily a problem, as long as the tilt axis and tilt<br>
angles are specified accordingly.<br>
<br>
Best wishes,<br>
<br>
--<br>
Ricardo Diogo Righetto<br>
<br>
<br>
Em seg., 18 de mar. de 2024 ?s 02:57, Sylvain Trepout <<br>
<a href="mailto:sylvain.trepout@monash.edu" target="_blank">sylvain.trepout@monash.edu</a>> escreveu:<br>
<br>
> Hi Zongli and Ricardo, and tomo enthusiasts,<br>
><br>
> Same here, on a Krios G4 with Falcon 4i and Tomo software version<br>
> 5.16.0.6389, the orientation of the tilt axis is not reported correctly.<br>
> TiltAxisAngle = -174.72<br>
> RotationAngle = 84.72<br>
><br>
> But the correct tilt axis angle found by IMOD is 95.07<br>
> It does not fit the formula you gave Ricardo though... off by just 10<br>
> degrees.<br>
> Or maybe this is a flip and then the orientation would be ok, but this<br>
> would change the handedness.<br>
><br>
> For comparison, on the Krios G1 (with a K3 and an older version of Tomo<br>
> software)<br>
> TiltAxisAngle = -174.47<br>
> RotationAngle = -84.72<br>
> The correct tilt axis orientation (as found by IMOD) is around 87 (this<br>
> fits your formula, Ricardo).<br>
><br>
> Best wishes,<br>
> Sylvain<br>
><br>
> PhD<br>
> Research Fellow - Microscopy Australia<br>
><br>
> *Monash University*<br>
> Ramaciotti Centre for Cryo-EM<br>
> G92, 15 Innovation Walk, Clayton Campus<br>
> Victoria, 3800 Australia<br>
><br>
> E: <a href="mailto:sylvain.trepout@monash.edu" target="_blank">sylvain.trepout@monash.edu</a><br>
><br>
><br>
> On Sun, 17 Mar 2024 at 07:53, Zongli Li <<a href="mailto:zongli.li@gmail.com" target="_blank">zongli.li@gmail.com</a>> wrote:<br>
><br>
>> Dear Ricardo,<br>
>><br>
>> Thank you so much! This makes a lot of sense.<br>
>> Best wishes,<br>
>><br>
>> Zongli<br>
>><br>
>> On Sat, Mar 16, 2024 at 12:37?PM Ricardo Righetto <<br>
>> <a href="mailto:ricardorighetto@gmail.com" target="_blank">ricardorighetto@gmail.com</a>> wrote:<br>
>><br>
>>> Dear Zongli,<br>
>>><br>
>>> Thanks for bringing this up. As far as I know this is a bug (?) in<br>
>>> Tomo5. This is how you convert the TiltAxisAngle from the Tomo5 mdoc to<br>
>>> match the IMOD/SerialEM convention (followed by RELION and other packages):<br>
>>><br>
>>> TiltAxisAngle_SerialEM = TiltAxisAngle_Tomo5 * (-1) - 90<br>
>>><br>
>>> Thanks to Grigory Sharov for reporting this conversion previously on the<br>
>>> EMAN2 mailing list:<br>
>>> <a href="https://urldefense.com/v3/__https://groups.google.com/g/eman2/c/piux0GdX_7M/m/ScQNh9TNAQAJ__;!!Mih3wA!Cm7XcxF6VqlPRi_nuhuhA5UJxZ1CPlAv8FHSp5bPNetfDBaUg5hUU07A7CuZSCG0fmFna5-2cySw_u-xToQzMzQ2IizT$" rel="noreferrer" target="_blank">https://urldefense.com/v3/__https://groups.google.com/g/eman2/c/piux0GdX_7M/m/ScQNh9TNAQAJ__;!!Mih3wA!Cm7XcxF6VqlPRi_nuhuhA5UJxZ1CPlAv8FHSp5bPNetfDBaUg5hUU07A7CuZSCG0fmFna5-2cySw_u-xToQzMzQ2IizT$</a> <br>
>>> <<a href="https://urldefense.com/v3/__https://groups.google.com/g/eman2/c/piux0GdX_7M/m/ScQNh9TNAQAJ__;!!Mih3wA!Go-c8e-QwJVBsU0qttyzgSDv-knvzqN6KJbTdN11SKI-RKgQ9dktWrwtqL_J9HC10IqJx5KxDD2MNvPZcrYB$" rel="noreferrer" target="_blank">https://urldefense.com/v3/__https://groups.google.com/g/eman2/c/piux0GdX_7M/m/ScQNh9TNAQAJ__;!!Mih3wA!Go-c8e-QwJVBsU0qttyzgSDv-knvzqN6KJbTdN11SKI-RKgQ9dktWrwtqL_J9HC10IqJx5KxDD2MNvPZcrYB$</a>><br>
>>><br>
>>> TiltAxisAngle and RotationAngle should always be 90 degrees apart in any<br>
>>> mdoc:<br>
>>> <a href="https://urldefense.com/v3/__https://bio3d.colorado.edu/SerialEM/hlp/html/about_formats.htm__;!!Mih3wA!Cm7XcxF6VqlPRi_nuhuhA5UJxZ1CPlAv8FHSp5bPNetfDBaUg5hUU07A7CuZSCG0fmFna5-2cySw_u-xToQzM_tT6y4J$" rel="noreferrer" target="_blank">https://urldefense.com/v3/__https://bio3d.colorado.edu/SerialEM/hlp/html/about_formats.htm__;!!Mih3wA!Cm7XcxF6VqlPRi_nuhuhA5UJxZ1CPlAv8FHSp5bPNetfDBaUg5hUU07A7CuZSCG0fmFna5-2cySw_u-xToQzM_tT6y4J$</a> <br>
>>> <<a href="https://urldefense.com/v3/__https://bio3d.colorado.edu/SerialEM/hlp/html/about_formats.htm__;!!Mih3wA!Go-c8e-QwJVBsU0qttyzgSDv-knvzqN6KJbTdN11SKI-RKgQ9dktWrwtqL_J9HC10IqJx5KxDD2MNiJtThea$" rel="noreferrer" target="_blank">https://urldefense.com/v3/__https://bio3d.colorado.edu/SerialEM/hlp/html/about_formats.htm__;!!Mih3wA!Go-c8e-QwJVBsU0qttyzgSDv-knvzqN6KJbTdN11SKI-RKgQ9dktWrwtqL_J9HC10IqJx5KxDD2MNiJtThea$</a>><br>
>>> That's why you got the "correct" result when using the value from<br>
>>> RotationAngle as the tilt axis. Beware though that you may have inverted<br>
>>> handedness in your reconstructions. In my experience, the conversion above<br>
>>> should give the correct results.<br>
>>><br>
>>> Would be great if others can comment if this is indeed the case, and why<br>
>>> this bug persists for so long...<br>
>>><br>
>>> Best wishes,<br>
>>><br>
>>> --<br>
>>> Ricardo Diogo Righetto<br>
>>><br>
>>><br>
>>> Em sex., 15 de mar. de 2024 ?s 21:33, Zongli Li <<a href="mailto:zongli.li@gmail.com" target="_blank">zongli.li@gmail.com</a>><br>
>>> escreveu:<br>
>>><br>
>>>> Dear colleagues,<br>
>>>><br>
>>>> We recently collected some tilt series data on vitrified ribosome<br>
>>>> sample using ThermoFisher Scientifc's Tomo5 software. The data were<br>
>>>> collected on Falcon 4i. When trying to process the data using the new<br>
>>>> version of relion tomo workflow (relion 5.0-beta), we are wondering what<br>
>>>> parameter we should use from the dmoc file for tilt axis angle<br>
>>>> (TiltAxisAngle or RotationAngle). If the TiltAxisAngle is used in our test<br>
>>>> run, the reconstructed tomogram is nonsense, but the reconstructed tomogram<br>
>>>> looks okay if RotationAngle is used. Any suggestions or explanations on<br>
>>>> this would be highly appreciated.<br>
>>>><br>
>>>> Best,<br>
>>>><br>
>>>> Zongli<br>
>>>> _______________________________________________<br>
>>>> 3dem mailing list<br>
>>>> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a><br>
>>>> <a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem" rel="noreferrer" target="_blank">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a><br>
>>>><br>
>>> _______________________________________________<br>
>> 3dem mailing list<br>
>> <a href="mailto:3dem@ncmir.ucsd.edu" target="_blank">3dem@ncmir.ucsd.edu</a><br>
>> <a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem" rel="noreferrer" target="_blank">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a><br>
>><br>
><br>
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