Re: [ccp4bb] About model building

[Kay Diederichs]

Hi Sam,

What you write does not sound good to me.
Did you have Phaser check "all possible [spacegroups] in same pointgroup" (this 
is how it is called in ccp4i)? P321 is quite rare, compared to P3121 and P3221. 
If you miss a screw axis, you will not find a solution.
An R-factor of 50% does not at all prove that the solution is correct.

Finally, an outer-shell CC1/2 of 0.9 means that the crystals diffract 
significantly higher than 1.9A (maybe 1.7A or even better).
You should use much more data from that data collection rather than truncating 
at 1.9A; even data from the corners of the detector are useful if there is 
diffraction recorded (with, say, I/sigma >1 or CC1/2>0.14 ). 
There exists no "completeness police" that would tell you "the completeness in 
your highest shell is less than 90% so you cannot use these data" or similar. 
In particular if you have little sequence knowledge for chain B, you want to 
maximize the information gained from the experiment; this helps the maps, the 
refinement, and therefore the sequence assignment. Every reflection carries 
some amount of information; that amount depends on how accurately it is 
measured.

If the detector edges were limiting the max resolution and you have more 
crystals, collect additional datasets at closer distance.

HTH,
Kay
 

On Mon, 6 Nov 2023 13:09:59 +0800, Sam Tang  wrote:

>Dear all
>
>Thanks for all the input. I will definitely try out in the coming couple of
>days. To provide more information:
>- the data was checked in Xtriage and no major pathology was found.
>Completeness was 99.2% with multiplicity of >13. Mean I/sigma(I) 18.8,
>CC1/2 for outer shell 0.902.
>- After one round of refinement, the R-factor was around 0.5, which looked
>reasonable given the incorrect B-chain was not removed and sequence
>deviations in chain A not yet rectified.
>- While I said Arp/warp failed to rebuild the model, it did return 3
>helices in chain A and one in chain B when I ran in the QuickFold mode
>(secondary structure tracing) later on. So I am going to start with the
>helix in chain B and see how further manual rebuild goes.
>
>Thanks again and I shall send an update for any progress.
>
>Kind regards
>
>Sam
>
>
>On Sun, 5 Nov 2023 at 06:26, Firdous Tarique 
>wrote:
>
>> Do the mass spec of your crystal to identify the other protein. Once done
>> solve your structure and build the complete model. This should be straight
>> forward and quick.
>>
>> Best Wishes
>>
>> On Sat, 4 Nov 2023, 09:05 Sam Tang,  wrote:
>>
>>> Dear community,
>>>
>>> I am solving the structure of a complex between proteins A and B, where A
>>> is a protein with known homologs and B is a novel protein isolated from
>>> plant. The diffraction data was at 1.9 Ang collected in-house, indexed to
>>> P321. Using A as the search model, we have got a reasonable solution where,
>>> after one round of refinement, the A chain fits the map pretty well. What's
>>> left was to extend the termini and fit a few rotamers.
>>>
>>> For protein B (B chain) I have tried the web version of ARP/wARP but the
>>> outcome was not really good. The model was not successfully built as
>>> indicated by low model completeness and score. The tricky thing may be that
>>> we do not have the complete sequence information of this protein B in-hand.
>>> (The other way round, we more or less wish to rely on the high resolution
>>> data to confirm its sequence.) What approach would you then recommend to
>>> build the B chain in this scenario?
>>>
>>> Thanks in advance and best regards,
>>>
>>> Sam
>>>
>>> --
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>>>
>>
>
>
>
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Re: [ccp4bb] Mean B value in refmac5

[Qixu Cai]

Thanks for your reply. Does the refmac5 program weight the mean B value by
the resolution?

Qixu Cai
Email: caiq...@gmail.com



Ian Tickle  于2023年11月5日周日 19:34写道：

> The arithmetic mean B value from the structure as quoted everywhere is
> pretty meaningless anyway and 10 Ang.^2 either way is probably not
> significant. Let's say some waters or LYS side-chains or whatever have B =
> 1000 Ang.^2. That will bias the mean B upwards, but those atoms do not
> contribute significantly to the total scattering except at very low
> resolution and might as well not be there, so they should not be included
> in the mean. A better method would be to weight the mean by the scattering
> power at the resolution limit. That should more closely match the B value
> from the Wilson plot.
>
> Cheers
>
> Ian
>
>
> On Sun, 5 Nov 2023, 10:45 Qixu Cai,  wrote:
>
>> Dear all,
>>
>> I found that the "Mean B value (OVERALL, A**2)" reported by refmac5 in
>> the head region of pdb file is 62.76. However, I calculated the average B
>> value of all B factors from all atoms and the result is 67.7. What makes
>> the difference? The canonical restrained refinement mode was used in
>> refmac5. When we prepare the Table 1 of manuscript, which one is correct
>> for the "average B value"?
>>
>> Thanks and best regards,
>> Qixu Cai
>> Email: caiq...@gmail.com
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>>
>



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Re: [ccp4bb] Mean B value in refmac5

[Qixu Cai]

I tried both and none of them is the same as reported by refmac5.

Qixu Cai
Email: caiq...@gmail.com



James Holton  于2023年11月6日周一 01:11写道：

> Are you averaging over all ATOM records?  Or are you including HETATM as
> well?
>
> On 11/5/2023 5:45 AM, Qixu Cai wrote:
>
> Dear all,
>
> I found that the "Mean B value (OVERALL, A**2)" reported by refmac5 in the
> head region of pdb file is 62.76. However, I calculated the average B value
> of all B factors from all atoms and the result is 67.7. What makes the
> difference? The canonical restrained refinement mode was used in refmac5.
> When we prepare the Table 1 of manuscript, which one is correct for the
> "average B value"?
>
> Thanks and best regards,
> Qixu Cai
> Email: caiq...@gmail.com
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
>
>



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Re: [ccp4bb] Mean B value in refmac5

[Qixu Cai]

Yes. I tried to weight those B values by occupancies, but the result is
still not the same as the value reported by refmac5.

Qixu Cai
Email: caiq...@gmail.com



Eleanor Dodson <176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
于2023年11月6日周一 14:04写道：

> Are there some atoms with occupancies < 1.0 ?
> You would need to weight those Bs by occ
>
> On Sun, 5 Nov 2023 at 11:34, Ian Tickle  wrote:
>
>> The arithmetic mean B value from the structure as quoted everywhere is
>> pretty meaningless anyway and 10 Ang.^2 either way is probably not
>> significant. Let's say some waters or LYS side-chains or whatever have B =
>> 1000 Ang.^2. That will bias the mean B upwards, but those atoms do not
>> contribute significantly to the total scattering except at very low
>> resolution and might as well not be there, so they should not be included
>> in the mean. A better method would be to weight the mean by the scattering
>> power at the resolution limit. That should more closely match the B value
>> from the Wilson plot.
>>
>> Cheers
>>
>> Ian
>>
>>
>> On Sun, 5 Nov 2023, 10:45 Qixu Cai,  wrote:
>>
>>> Dear all,
>>>
>>> I found that the "Mean B value (OVERALL, A**2)" reported by refmac5 in
>>> the head region of pdb file is 62.76. However, I calculated the average B
>>> value of all B factors from all atoms and the result is 67.7. What makes
>>> the difference? The canonical restrained refinement mode was used in
>>> refmac5. When we prepare the Table 1 of manuscript, which one is correct
>>> for the "average B value"?
>>>
>>> Thanks and best regards,
>>> Qixu Cai
>>> Email: caiq...@gmail.com
>>>
>>>
>>> --
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>>
>
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>
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Re: [ccp4bb] About model building

[Boaz Shaanan]

Hi,
If you still have crystals left, you could soak crystals with KI3 and collect 
data at Cu wavelength for SAD phasing, which could help you to resolve the 
missing piece. Maybe.
Cheers,
Boaz

Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben Gurion University
Beer Sheva, Israel

On Nov 4, 2023 10:04, Sam Tang  wrote:
Dear community,

I am solving the structure of a complex between proteins A and B, where A is a 
protein with known homologs and B is a novel protein isolated from plant. The 
diffraction data was at 1.9 Ang collected in-house, indexed to P321. Using A as 
the search model, we have got a reasonable solution where, after one round of 
refinement, the A chain fits the map pretty well. What's left was to extend the 
termini and fit a few rotamers.

For protein B (B chain) I have tried the web version of ARP/wARP but the 
outcome was not really good. The model was not successfully built as indicated 
by low model completeness and score. The tricky thing may be that we do not 
have the complete sequence information of this protein B in-hand. (The other 
way round, we more or less wish to rely on the high resolution data to confirm 
its sequence.) What approach would you then recommend to build the B chain in 
this scenario?

Thanks in advance and best regards,

Sam



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[ccp4bb] Structural Biology Facility Manager vacancy at the John Innes Centre, Norwich UK

[David Lawson (JIC)]

Dear All,

We have a vacancy for a Structural Biology Facility Manager at the John Innes 
Centre.

The John Innes Centre is an independent, international centre of excellence in 
plant and microbial sciences. We nurture a creative, curiosity-led approach to 
answering important questions in bioscience, and translate that knowledge into 
societal benefits. Recent investment from the UKRI Infrastructure Fund will 
ensure the JIC's ability to contribute cutting-edge, world-leading research 
into the future, underpinned by excellent facilities. We are an equal 
opportunities employer, actively supporting inclusivity and diversity, and we 
provide support for ongoing career development.

We are looking for someone with extensive postdoctoral expertise in protein 
crystallography; experience of cryo-EM and SAXS would also be advantageous. The 
successful applicant will oversee the Facility and contribute to a wide variety 
of projects. The post holder will be expected to provide training and to ensure 
financial sustainability of the Facility though usage recharges and funding 
applications.

Closing date - 3rd December 2023.
Salary - £43,550 - £54,900
Contract - indefinite, full-time

For further details, please visit our website http://jobs.jic.ac.uk or contact 
the Human Resources team on 01603 450814 or nbi.recruitm...@nbi.ac.uk quoting 
reference 1004561.

Best Wishes,

Dave Lawson

---

Prof. David M. Lawson
Department of Biochemistry and Metabolism,
John Innes Centre,
Norwich,
NR4 7UH, UK.
Tel: +44-(0)1603-450725
Web: https://www.jic.ac.uk/people/david-lawson
Email: david.law...@jic.ac.uk





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[ccp4bb] Bob Blessing

[Michael Malkowski]

It is with much sadness that I write to inform the community that Robert (Bob) 
Blessing passed away on October 30th.

Blessing graduated from King’s College in 1962. He received a Ph.D. in 
Chemistry from Ohio University, and in the following four years, he carried out 
post-doctoral studies at the University of Lund, Sweden; the University of 
Pittsburgh; and two at the State University of New York at Buffalo.

After working as an Assistant Professor of Chemistry at Mercyhurst College in 
Erie, Pennsylvania for four years, Blessing moved to Buffalo in 1978, where he 
worked as a Research Scientist at Hauptman-Woodward Medical Research Institute 
(HWI), formerly the Medical Foundation of Buffalo. At HWI, he worked with Dr. 
G. David Smith, Emeritus HWI Researcher, publishing papers on the crystal 
structure of human insulin. Blessing also developed algorithms and software for 
crystallographic data reduction and error analysis that are widely used in 
laboratories worldwide. He was an active member of the American 
Crystallographic Association and served as President of the Pittsburgh 
Diffraction Society.

In 1991, Blessing was appointed as an Associate Professor of Chemistry at the 
University of Buffalo (UB) while continuing his duties at HWI. In 2001, 
Blessing designed the graduate studies program for the newly formed Department 
of Structural Biology within the Jacobs School of Medicine and Biomedical 
Sciences at UB. He was subsequently promoted to Professor within the Department 
and later served as Director of Graduate Studies and Chair. Blessing was an 
active member in the Department until his retirement in 2021.

Blessing presented work at national and international conferences. He 
coauthored many publications, including with Herbert Hauptman, the 1985 Nobel 
Prize winner in Chemistry. During four different periods, he was a visiting 
professor in Nancy, France, at the Université de Lorraine where he co-authored 
ten publications with Claude Lecomte, Professor of Crystallography and Physics.

Colleague Jane Griffin, Principal Research Scientist at HWI, described him as 
“a trusted friend and colleague; always a kind and loving presence… An 
outstanding scientist, teacher, scholar, Father, friend, colleague… Never dull, 
full of wit, and never mean-spirited.”

He will be truly missed.


--
Michael G. Malkowski, 
Ph.D.
Professor and Chair
Department of Structural Biology
Jacobs School of Medicine and Biomedical Sciences
University at Buffalo
955 Main Street, Room 5154
Buffalo, New York 14203-1121
office: 716-829-3698
email: mg...@buffalo.edu
twitter: @ub_malkowski



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[ccp4bb] RES: [ccp4bb] About model building

[Rafael Marques]

Hi Sam.

If you still have any of your crystals or any protein solution left in the well 
you harvested your crystals, I would run a MS/MS with them. Next step would be 
to run AF with your known chain A and your best Mass Spec hit (s), and use the 
resulting model for MR.

Good luck


Rafael Marques da Silva
PhD Student – Structural Biology
University of Leicester

Mestre em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"



De: CCP4 bulletin board  em nome de Boaz Shaanan 

Enviado: Monday, November 6, 2023 2:41:43 PM
Para: CCP4BB@JISCMAIL.AC.UK 
Assunto: Re: [ccp4bb] About model building

Hi,
If you still have crystals left, you could soak crystals with KI3 and collect 
data at Cu wavelength for SAD phasing, which could help you to resolve the 
missing piece. Maybe.
Cheers,
Boaz

Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben Gurion University
Beer Sheva, Israel

On Nov 4, 2023 10:04, Sam Tang  wrote:
Dear community,

I am solving the structure of a complex between proteins A and B, where A is a 
protein with known homologs and B is a novel protein isolated from plant. The 
diffraction data was at 1.9 Ang collected in-house, indexed to P321. Using A as 
the search model, we have got a reasonable solution where, after one round of 
refinement, the A chain fits the map pretty well. What's left was to extend the 
termini and fit a few rotamers.

For protein B (B chain) I have tried the web version of ARP/wARP but the 
outcome was not really good. The model was not successfully built as indicated 
by low model completeness and score. The tricky thing may be that we do not 
have the complete sequence information of this protein B in-hand. (The other 
way round, we more or less wish to rely on the high resolution data to confirm 
its sequence.) What approach would you then recommend to build the B chain in 
this scenario?

Thanks in advance and best regards,

Sam



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Re: [ccp4bb] RES: [ccp4bb] About model building

[Sam Tang]

Dear all

Thanks again for more input to the question. And credits to Eleanor and Kay
for pointing out the high R-factor and possible issue with the space group.
Their advice prompted me to revisit the MR solution and it happens that
another solution, in P3121, gave a better map with R-factor after one round
of refinement being 0.33/0.36, which was a remarkable difference with the
P321 solution (R~0.5).

So the next step I would take would be to re-try Arp/warp and see if things
work out with poly-A. I shall update the community with the outcome.

Kind regards

Sam




On Tue, 7 Nov 2023 at 01:13, Rafael Marques 
wrote:

> Hi Sam.
>
>
>
> If you still have any of your crystals or any protein solution left in the
> well you harvested your crystals, I would run a MS/MS with them. Next step
> would be to run AF with your known chain A and your best Mass Spec hit (s),
> and use the resulting model for MR.
>
>
>
> Good luck
>
>
>
>
> Rafael Marques da Silva
>
> PhD Student – Structural Biology
>
> University of Leicester
>
>
>
> Mestre em Física Biomolecular
>
> Universidade de São Paulo
>
>
>
> Bacharel em Ciências Biológicas
>
> Universidade Federal de São Carlos
>
>
>
> phone: +55 16 99766-0021
>
>
>
> *   "A sorte acompanha uma mente bem treinada"*
>
> **
>
>
> --
> *De:* CCP4 bulletin board  em nome de Boaz Shaanan
> 
> *Enviado:* Monday, November 6, 2023 2:41:43 PM
> *Para:* CCP4BB@JISCMAIL.AC.UK 
> *Assunto:* Re: [ccp4bb] About model building
>
> Hi,
> If you still have crystals left, you could soak crystals with KI3 and
> collect data at Cu wavelength for SAD phasing, which could help you to
> resolve the missing piece. Maybe.
> Cheers,
> Boaz
>
> Boaz Shaanan, Ph.D.
> Dept. of Life Sciences
> Ben Gurion University
> Beer Sheva, Israel
>
> On Nov 4, 2023 10:04, Sam Tang  wrote:
> Dear community,
>
> I am solving the structure of a complex between proteins A and B, where A
> is a protein with known homologs and B is a novel protein isolated from
> plant. The diffraction data was at 1.9 Ang collected in-house, indexed to
> P321. Using A as the search model, we have got a reasonable solution where,
> after one round of refinement, the A chain fits the map pretty well. What's
> left was to extend the termini and fit a few rotamers.
>
> For protein B (B chain) I have tried the web version of ARP/wARP but the
> outcome was not really good. The model was not successfully built as
> indicated by low model completeness and score. The tricky thing may be that
> we do not have the complete sequence information of this protein B in-hand.
> (The other way round, we more or less wish to rely on the high resolution
> data to confirm its sequence.) What approach would you then recommend to
> build the B chain in this scenario?
>
> Thanks in advance and best regards,
>
> Sam
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
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>
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Re: [ccp4bb] RES: [ccp4bb] About model building

[Oganesyan, Vaheh]

You may as well have fun by manually building your molecule B into 1.9A ed map 
when R-factors are already in mid 30s.

Vaheh

From: CCP4 bulletin board  On Behalf Of Sam Tang
Sent: Monday, November 6, 2023 2:02 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] RES: [ccp4bb] About model building

Dear all

Thanks again for more input to the question. And credits to Eleanor and Kay for 
pointing out the high R-factor and possible issue with the space group. Their 
advice prompted me to revisit the MR solution and it happens that another 
solution, in P3121, gave a better map with R-factor after one round of 
refinement being 0.33/0.36, which was a remarkable difference with the P321 
solution (R~0.5).

So the next step I would take would be to re-try Arp/warp and see if things 
work out with poly-A. I shall update the community with the outcome.

Kind regards

Sam



On Tue, 7 Nov 2023 at 01:13, Rafael Marques 
mailto:rafael_mmsi...@hotmail.com>> wrote:
Hi Sam.

If you still have any of your crystals or any protein solution left in the well 
you harvested your crystals, I would run a MS/MS with them. Next step would be 
to run AF with your known chain A and your best Mass Spec hit (s), and use the 
resulting model for MR.

Good luck


Rafael Marques da Silva
PhD Student – Structural Biology
University of Leicester

Mestre em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"



De: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
em nome de Boaz Shaanan mailto:bshaa...@bgu.ac.il>>
Enviado: Monday, November 6, 2023 2:41:43 PM
Para: CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Assunto: Re: [ccp4bb] About model building

Hi,
If you still have crystals left, you could soak crystals with KI3 and collect 
data at Cu wavelength for SAD phasing, which could help you to resolve the 
missing piece. Maybe.
Cheers,
Boaz

Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben Gurion University
Beer Sheva, Israel

On Nov 4, 2023 10:04, Sam Tang 
mailto:samtys0...@gmail.com>> wrote:
Dear community,

I am solving the structure of a complex between proteins A and B, where A is a 
protein with known homologs and B is a novel protein isolated from plant. The 
diffraction data was at 1.9 Ang collected in-house, indexed to P321. Using A as 
the search model, we have got a reasonable solution where, after one round of 
refinement, the A chain fits the map pretty well. What's left was to extend the 
termini and fit a few rotamers.

For protein B (B chain) I have tried the web version of ARP/wARP but the 
outcome was not really good. The model was not successfully built as indicated 
by low model completeness and score. The tricky thing may be that we do not 
have the complete sequence information of this protein B in-hand. (The other 
way round, we more or less wish to rely on the high resolution data to confirm 
its sequence.) What approach would you then recommend to build the B chain in 
this scenario?

Thanks in advance and best regards,

Sam



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Re: [ccp4bb] problem with phenix in macos sonoma 14.1

[Nigel Moriarty]

Debipreeta

There is a fix with instructions on the download page and it is also here
on the Phenix bulletin board (
https://phenix-online.org/pipermail/phenixbb/2023-October/025579.html).

Cheers

Nigel

---
Nigel W. Moriarty
Building 33R0349, Molecular Biophysics and Integrated Bioimaging
Lawrence Berkeley National Laboratory
Berkeley, CA 94720-8235
Email : nwmoria...@lbl.gov
Web  : CCI.LBL.gov
ORCID : orcid.org/-0001-8857-9464


On Mon, Nov 6, 2023 at 2:25 PM debipreeta bhowmik <
debipreetabhow...@gmail.com> wrote:

> I have updated the mac (macos sonoma 14.1. ).
> When I am trying to open Phenix it is showing another session is running
> and I am not able to reopen it by clicking yes also.
> I attached the message.
> Can you please tell me what to do?
>
> Regards,
> Debipreeta Bhowmik
> Assistant Academic Research Scientist
> Emory University
> United States
>
> --
>
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[ccp4bb] Biochemist post-doctoral associate position

[O'Neill, Hugh]

Biochemist Position at Oak Ridge National Laboratory


We are seeking a Postdoctoral Research Associate with expertise in biochemistry 
and molecular biology to join an interdisciplinary team in the Neutron 
Scattering Division (NSD) at Oak Ridge National Laboratory. You will contribute 
to a project that aims to develop an integrated platform for rapid production 
and characterization of viral proteins to aid in the design of therapeutics. 
You will be responsible for developing and executing approaches to produce 
deuterium-labeled proteins for small-angle neutron scattering measurements to 
provide insights into the structural properties of these macromolecular 
complexes. Taking this position, you will have the opportunity to work with a 
team comprising of specialists in structural biology, assay development, X-ray 
and neutron diffraction and scattering, and computation. As part of our 
research team, you will be affiliated with the ORNL Center for Structural 
Molecular Biology, and you will have access 
to a tool suite that includes leading small-angle scattering and neutron 
diffraction facilities, bio-fermentation laboratories, and biophysical 
characterization laboratories, in addition to in-house small angle X-ray 
scattering and X-ray diffraction instrumentation. The position represents an 
excellent opportunity for you to develop your career and interact with leading 
scientists from around the world.

A link to the position advertisement can be found here:

https://jobs.ornl.gov/job/Oak-Ridge-Postdoctoral-Research-Associate-Biochemist-TN-37830/1081996500/






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