[ccp4bb] X-ray crystallography defense questions

[Jessica Besaw]

Hello CCP4!

I am preparing for my PhD defense on X-ray crystallography of membrane
proteins.

So my ask from you folks is: *What X-ray crystallography questions would
you ask in a PhD defense? *

All questions - easy, tough, tricky - are welcome.

Thank you kindly,

Jessica Besaw



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Re: [ccp4bb] A question of density

[Jessica Besaw]

Hello Matthias,

Excellent point. Most of the the ordered water are easily visible at 2
rmsd. The central disordered (or partially occupied) water becomes visible
only at 1.3 - 1.4 rmsd, and it is very visible at 1 rmsd (which I have
displayed all of the maps). In your opinion, do you think this would be
noise?

Jessica

On Wed, 4 Mar 2020 at 12:45, Barone, Matthias  wrote:

> hey Jessica
>
> a tip that might come up later on anyway: once you put every reasonable
> bit into the desity, what I like to to when facing such blobbs: I take a
> well defined water out to create a diff density at a position where I know
> it is real. Having a feeling of how much you have to contour the diff
> density at that point can give you a good feeling how much of noise is
> actually in your density right in between the waters..?
>
> best, matthias
>
>
> Dr. Matthias Barone
>
> AG Kuehne, Rational Drug Design
>
> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> Robert-Rössle-Strasse 10
> 13125 Berlin
>
> Germany
> Phone: +49 (0)30 94793-284
> ----------
> *From:* CCP4 bulletin board  on behalf of Jessica
> Besaw 
> *Sent:* Wednesday, March 4, 2020 6:42:34 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] A question of density
>
> Hey Nukri,
>
> Here are the details: Rwork/Rfree = 0.21 / 0.23 for a 2 Angstrom structure
>
> I absolutely agree with you on the refinement. I did previously do that,
> and I attached the picture.
>
> What is the BB?
>
> Cheers!
>
> Jessica
>
>
>
>
>
>
> On Wed, 4 Mar 2020 at 12:20, Nukri Sanishvili  wrote:
>
>> Hi Jessica,
>> You do not say how well is the rest of the structure refined.
>> First, you should refine the structure best you can, without placing
>> anything in the unclear blob of your interest so to obtain the best
>> possible phases and hopefully improve the blob density as well.
>> Then you should let the BB see what that density looks like. Looking at
>> only the list of possibilities has very little value without seeing the
>> density itself.
>> Best wishes,
>> Nukri
>>
>> On Wed, Mar 4, 2020 at 11:10 AM Jessica Besaw 
>> wrote:
>>
>>> Hello friends,
>>>
>>> I have a "blob" of density in an active site of my protein.
>>>
>>> I am struggling to determine if I should place a water in this spot, if
>>> I should model it as a disordered water, if the density may be a ligand
>>> that I have not considered, or if it should be left as unaccounted for
>>> density. I would like to publish this structure without compromising the
>>> science.
>>>
>>> I have attached several possibilities that I have considered below.
>>>
>>> Any suggestions would be appreciated.
>>>
>>> Cheers!
>>>
>>> Jessica Besaw
>>>
>>>
>>>
>>> --
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>>
>>
> --
>
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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] 1 out of 2 proteins in asymmetric unit does not fit density

[Jessica Besaw]

THE PROBLEM IS SOLVED!

Thank you all for you suggestions. I applied all the suggestion across all
datasets collected (not just the one shown above).

The error & solution: Incorrect space group assignment (new space group P
21 2 21) and ignoring tNCS.

This is the BEST Christmas gift a crystallographer could get.  Thank you
everyone!

Cheers!

Jessica












On Mon, 16 Dec 2019 at 16:16, Wim Burmeister  wrote:

> Hello,
> I would guess that the badly fitting molecule may be upside down (related
> my an 2-fold axis).
> I would use the first, partially refined structure for another round of
> molecular replacement in P212121 with molrep, using the model as well as a
> partial solution as as asearch model.
> The translational self peak in the native Patterson may be misleading. I
> came recently across a similar problem.
> Regards
> Wim
>
> ------
> *De: *"Jessica Besaw" 
> *À: *"CCP4BB" 
> *Envoyé: *Lundi 16 Décembre 2019 20:29:38
> *Objet: *Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] 1 out of 2 proteins in
> asymmetric unit does not fit density
>
> There have been two potential space groups:
> P212121 - Rfree = 36%
> P21212 - Rfree = 45%
>
> Xtriage reports that twinning is unlikely.
>
> Cheers!
>
> Jessica
>
>
>
>
>
> On Mon, 16 Dec 2019 at 13:56, Jürgen Bosch  wrote:
>
>> What’s your spacegroup ? RWork / RFree?
>> Twinning by any chance?
>>
>> Jürgen
>>
>> __
>> Jürgen Bosch, Ph.D.
>> Division of Pediatric Pulmonology and Allergy/Immunology
>> Case Western Reserve University
>> 2109 Adelbert Rd, BRB 835
>> Cleveland, OH 44106
>> Phone: 216.368.7565
>> Fax: 216.368.4223
>> https://www.linkedin.com/in/jubosch/
>>
>> CEO & Co-Founder at InterRayBio, LLC
>>
>> Johns Hopkins University
>> Bloomberg School of Public Health
>> Department of Biochemistry & Molecular Biology
>>
>> On Dec 16, 2019, at 1:50 PM, Jessica Besaw  wrote:
>>
>> I am crystallizing this membrane protein in a medium (bicelles) that
>> forms lamella like sheets that stack on top of each other.
>> The layer packing is shown below. Is this structure unreasonable?
>>
>> 
>>
>> On Mon, 16 Dec 2019 at 13:38, Reza Khayat  wrote:
>>
>>> Hi Jessica,
>>>
>>>
>>> The gap between the two proteins is a bit troubling. Perhaps it's the
>>> image, but why would a crystal form if there is no crystal contact between
>>> the two proteins?
>>>
>>>
>>> Reza
>>>
>>>
>>> Reza Khayat, PhD
>>> Assistant Professor
>>> City College of New York
>>> Department of Chemistry
>>> New York, NY 10031
>>> --
>>> *From:* CCP4 bulletin board  on behalf of Ashish
>>> Kumar 
>>> *Sent:* Monday, December 16, 2019 1:24 PM
>>> *To:* CCP4BB@JISCMAIL.AC.UK
>>> *Subject:* [EXTERNAL] Re: [ccp4bb] 1 out of 2 proteins in asymmetric
>>> unit does not fit density
>>>
>>> Hi Jessica,
>>>
>>> It may be possible because of wrong MR solution as well. How were your
>>> stats after MR.
>>> Also it is correct that it could be possible because of wrong space
>>> group.
>>> Try changing the Space group and repeat MR.
>>>
>>> Best Regards
>>> Ashish
>>>
>>> On 16 Dec 2019 22:56, "Jessica Besaw"  wrote:
>>>
>>> Dear community,
>>>
>>> I am having a lot of trouble solving a protein structure. I think my
>>> problem may caused by incorrectly placed proteins in molecular replacement.
>>> I have two proteins in my asymmetric unit. It appears that one protein fits
>>> perfectly, and the other one has many errors. (See snapshots below). I have
>>> tried deleting the parts of the protein (and even the whole protein) to try
>>> and rebuild it in COOT, but it was a bit too difficult for me to solve.
>>>
>>> I would appreciate any and all suggestions for potential strategies
>>> moving forward.
>>>
>>> Other information:
>>> (1) 2.4 Angstrom
>>> (2) 99% complete
>>> (3) "Translational NCS may be present at a level that may complicate
>>> refinement"
>>>
>>> Cheers!
>>>
>>> Jessica
>>>
>>> 
>>> 
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>

Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] 1 out of 2 proteins in asymmetric unit does not fit density

[Jessica Besaw]

There have been two potential space groups:

P212121 - Rfree = 36%
P21212 - Rfree = 45%

Xtriage reports that twinning is unlikely.

Cheers!

Jessica





On Mon, 16 Dec 2019 at 13:56, Jürgen Bosch  wrote:

> What’s your spacegroup ? RWork / RFree?
>
> Twinning by any chance?
>
> Jürgen
>
> __
> Jürgen Bosch, Ph.D.
> Division of Pediatric Pulmonology and Allergy/Immunology
> Case Western Reserve University
> 2109 Adelbert Rd, BRB 835
> Cleveland, OH 44106
> Phone: 216.368.7565
> Fax: 216.368.4223
> https://www.linkedin.com/in/jubosch/
>
> CEO & Co-Founder at InterRayBio, LLC
>
> Johns Hopkins University
> Bloomberg School of Public Health
> Department of Biochemistry & Molecular Biology
>
> On Dec 16, 2019, at 1:50 PM, Jessica Besaw  wrote:
>
> I am crystallizing this membrane protein in a medium (bicelles) that forms
> lamella like sheets that stack on top of each other.
>
> The layer packing is shown below. Is this structure unreasonable?
>
> 
>
> On Mon, 16 Dec 2019 at 13:38, Reza Khayat  wrote:
>
>> ​​Hi Jessica,
>>
>>
>> The gap between the two proteins is a bit troubling. Perhaps it's the
>> image, but why would a crystal form if there is no crystal contact between
>> the two proteins?
>>
>>
>> Reza
>>
>>
>> Reza Khayat, PhD
>> Assistant Professor
>> City College of New York
>> Department of Chemistry
>> New York, NY 10031
>> --
>> *From:* CCP4 bulletin board  on behalf of Ashish
>> Kumar 
>> *Sent:* Monday, December 16, 2019 1:24 PM
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* [EXTERNAL] Re: [ccp4bb] 1 out of 2 proteins in asymmetric
>> unit does not fit density
>>
>> Hi Jessica,
>>
>> It may be possible because of wrong MR solution as well. How were your
>> stats after MR.
>> Also it is correct that it could be possible because of wrong space group.
>> Try changing the Space group and repeat MR.
>>
>> Best Regards
>> Ashish
>>
>> On 16 Dec 2019 22:56, "Jessica Besaw"  wrote:
>>
>> Dear community,
>>
>> I am having a lot of trouble solving a protein structure. I think my
>> problem may caused by incorrectly placed proteins in molecular replacement.
>> I have two proteins in my asymmetric unit. It appears that one protein fits
>> perfectly, and the other one has many errors. (See snapshots below). I have
>> tried deleting the parts of the protein (and even the whole protein) to try
>> and rebuild it in COOT, but it was a bit too difficult for me to solve.
>>
>> I would appreciate any and all suggestions for potential strategies
>> moving forward.
>>
>> Other information:
>> (1) 2.4 Angstrom
>> (2) 99% complete
>> (3) "Translational NCS may be present at a level that may complicate
>> refinement"
>>
>> Cheers!
>>
>> Jessica
>>
>> 
>> 
>>
>>
>>
>>
>>
>>
>>
>> --
>>
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[ccp4bb] higher older tNCS

[Jessica Besaw]

Hello everyone,

Does anyone know of a good phenix tutorial on how to deal with higher order
translational non-crystallographic symmetry (tNCS) in a structure?

Ultimately, I need to know how to set TNCS NMOL to 3 in phaser of Phenix. I
am using the phenix GUI, and I am uncertain how to alter this parameter.

Any help would be greatly appreciated

Cheers!

Jessica




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