Re: [ccp4bb] Best method for carbohydrate refinement

[Bärbel Blaum]

Depending on your resolution or the quality of the local sugar density  
you also may want to give the GLYCAM server by Rob Woods' group a try.  
It includes a straightforward glycan builder, so no format issues, and  
does a great job in finding the lowest energy conformation for complex  
carbs, usually a good starting point for real space refinement if the  
density is not good enough for the beautiful carb module in COOT (or  
just for comparison, if you are curious).


Bärbel

Zitat von "Briggs, David C" :

The latest versions of COOT have a nifty carbohydrate building  
module for N-linked glycans, and Buccaneer can do carbohydrate  
validation  ( see appropriate  manuals ).


This article also highlights some of the issues and pitfalls you  
might encounter.


https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297920/

HTH

Dave

--

Dr David C Briggs

Hohenester Lab

Department of Life Sciences

Imperial College London

UK

http://about.me/david_briggs




From: CCP4 bulletin board  on behalf of  
Zhijie Li 

Sent: Friday, July 28, 2017 8:59:00 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Best method for carbohydrate refinement

Hi Gustavo,

If I understand you correctly, you are concerned about N-glycans
(N-glycosylation) on your proteins. According to your description, you
have 2 protein molecules in each ASU, each bearing one potential
N-glycan site. Then there are only two N-glycan sites you need to build
for each dataset ( I suppose you are not going to deposit everyone of
them? ). In most cases we do not see much ordered part of the N-glycans,
usually just one or both of the core GlcNac (NAG) residues . If this is
the case then it is not much work.

In very lucky cases you may see one or two rather complete N-glycans. I
think insect cells produce mostly pauci-mannose N-glycans (mostly
Man3-GlcNac2, less frequently Man4-GlcNac2) possibly carrying
core-fucosylation on the innermost GlcNAc.  Note that although mammals
only have core 1,6 fucose, insects may also have an additional core 1,3
fucose, discussed and illustrated in the following papers:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589692/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647355/

I am not aware of an automated procedure for building complex glycans
such as the N-glycans, so take extreme care making the correct linkages
if you have to build more than the two core NAGs. The Man3Gn2-Asn should
be
Man_alpha1,3-[Man_alpha1,6]-Man_beta1,4-GlcNac_beta1,4-GlcNAc_beta-Asn.
The core fucose(s), if present, will be alpha1,6- or alpha1,3- linked to
the innermost GlcNAc. Note that the core alpha1,3 fucose modification
occurs only when the core alpha1,6 is already there.


Zhijie


On 28/07/2017 1:43 PM, Gustavo Machado Alvares De Lima wrote:

Hello everybody,

I was looking for suggestion on the best way to identify and refine  
carbohydrates in a protein. This is the scenario:


- I have 10 molecules in the biological assembly
- I used sf9 expression system, so I have random glycolisation and  
types of glycolisations. I have some hint about possible  
glycolisation sites

- I believe I have only 1 glysolisation per monomer
- I have 2 molecule per AU. Space group C2


I would like (even if I have to write down a script for it) to  
track this glycolisation, identify the most probable ones,  
determine the restrictions for each and refine them. I could do it  
by hand on every refinement cycle (or a couple of cycles), but for  
many datasets it would take ages.


What protocol would you suggest?

I really appreciate any help in this subject.

Regards,
Gustavo




--
Bärbel Blaum, Ph.D.
Interfakultäres Institut für Biochemie (IFIB)
Hoppe-Seyler-Strasse 4
D-72076 Tübingen
Germany
+49 70 71 29 75 359

Re: [ccp4bb] Coot no GUI

[William G. Scott]

coot --no-graphics



William G. Scott

http://scottlab.ucsc.edu

> On Jul 28, 2017, at 1:34 PM, Reza Khayat  wrote:
> 
> Hi,
>  
> Is it possible to run Coot without the use of GUI?  We have some Coot scripts 
> for a pipeline and we’d like to do without the GUI. Thanks.
>  
> Best wishes,
> Reza
>  
> Reza Khayat, PhD
> Assistant Professor
> Department of Chemistry
> City College of New York
> 85 Saint Nicholas Terrace, CDI 2.318
> New York, NY 10031
> http://www.khayatlab.org/
> 212-650-6070

[ccp4bb] Coot no GUI

[Reza Khayat]

Hi,

Is it possible to run Coot without the use of GUI?  We have some Coot scripts 
for a pipeline and we'd like to do without the GUI. Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
85 Saint Nicholas Terrace, CDI 2.318
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

Re: [ccp4bb] Best method for carbohydrate refinement

[Zhijie Li]

Hi Gustavo,

If I understand you correctly, you are concerned about N-glycans 
(N-glycosylation) on your proteins. According to your description, you 
have 2 protein molecules in each ASU, each bearing one potential 
N-glycan site. Then there are only two N-glycan sites you need to build 
for each dataset ( I suppose you are not going to deposit everyone of 
them? ). In most cases we do not see much ordered part of the N-glycans, 
usually just one or both of the core GlcNac (NAG) residues . If this is 
the case then it is not much work.


In very lucky cases you may see one or two rather complete N-glycans. I 
think insect cells produce mostly pauci-mannose N-glycans (mostly 
Man3-GlcNac2, less frequently Man4-GlcNac2) possibly carrying 
core-fucosylation on the innermost GlcNAc.  Note that although mammals 
only have core 1,6 fucose, insects may also have an additional core 1,3 
fucose, discussed and illustrated in the following papers:


https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589692/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647355/

I am not aware of an automated procedure for building complex glycans 
such as the N-glycans, so take extreme care making the correct linkages 
if you have to build more than the two core NAGs. The Man3Gn2-Asn should 
be 
Man_alpha1,3-[Man_alpha1,6]-Man_beta1,4-GlcNac_beta1,4-GlcNAc_beta-Asn. 
The core fucose(s), if present, will be alpha1,6- or alpha1,3- linked to 
the innermost GlcNAc. Note that the core alpha1,3 fucose modification 
occurs only when the core alpha1,6 is already there.



Zhijie


On 28/07/2017 1:43 PM, Gustavo Machado Alvares De Lima wrote:

Hello everybody,

I was looking for suggestion on the best way to identify and refine 
carbohydrates in a protein. This is the scenario:

- I have 10 molecules in the biological assembly
- I used sf9 expression system, so I have random glycolisation and types of 
glycolisations. I have some hint about possible glycolisation sites
- I believe I have only 1 glysolisation per monomer
- I have 2 molecule per AU. Space group C2


I would like (even if I have to write down a script for it) to track this 
glycolisation, identify the most probable ones, determine the restrictions for 
each and refine them. I could do it by hand on every refinement cycle (or a 
couple of cycles), but for many datasets it would take ages.

What protocol would you suggest?

I really appreciate any help in this subject.

Regards,
Gustavo

[ccp4bb] Best method for carbohydrate refinement

[Gustavo Machado Alvares De Lima]

Hello everybody, 

I was looking for suggestion on the best way to identify and refine 
carbohydrates in a protein. This is the scenario:

- I have 10 molecules in the biological assembly
- I used sf9 expression system, so I have random glycolisation and types of 
glycolisations. I have some hint about possible glycolisation sites
- I believe I have only 1 glysolisation per monomer
- I have 2 molecule per AU. Space group C2


I would like (even if I have to write down a script for it) to track this 
glycolisation, identify the most probable ones, determine the restrictions for 
each and refine them. I could do it by hand on every refinement cycle (or a 
couple of cycles), but for many datasets it would take ages. 

What protocol would you suggest?

I really appreciate any help in this subject.

Regards, 
Gustavo

[ccp4bb] Position available

[Jeffrey B Bonanno]

Dear all:

Please see the advert below regarding an exciting position which is available 
at the Albert Einstein College of Medicine in New York City:

The laboratory of Tyler Grove and Steven Almo at the Albert Einstein College of 
Medicine seeks a highly motivated and innovative post-doctoral scientist 
interested in the structural, mechanistic and functional analysis of 
oxygen-sensitive biological macromolecules. Albert Einstein offers a unique 
environment for these studies, including participation in a multi-institutional 
consortium focused on enzyme functional annotation and metabolism discovery, 
along with one-of-a-kind structural genomics resources for high-throughput 
anaerobic purification, biochemical studies, and high resolution structural 
analyses. Successful candidates must have demonstrated considerable expertise 
in biological chemistry, with particular emphasis on protein chemistry. This 
position requires a significant level of independence, as well as the ability 
to interface with a larger team of investigators, and will afford extensive 
freedom in defining future courses of study.

Applicants must have a PhD, MD, or equivalent degree. Qualified applicants 
should submit a cover letter, curriculum vitae, and a brief summary of research 
accomplishments and goals in a single PDF document to:

steve.almo  einstein.yu.edu

and

tyler.grove  einstein.yu.edu

Applicants should also arrange to have three letters of reference sent directly 
to these email addresses.

Best,
Jeff

Jeffrey B. Bonanno, Ph.D.
Department of Biochemistry
Albert Einstein College of Medicine
1300 Morris Park Avenue
Bronx, NY 10461

Re: [ccp4bb] weird diffraction pattern

[Sanishvili, Ruslan]

Hi Chenjun Tang,


I have not followed the original discussion, so my apologies if I am repeating 
the device already given to you.

Crystallization optimization is always a good idea - nothing beats good quality 
crystals. In addition,

you should try collecting few images, especially in the "bad" orientation of 
the crystal, at ambient temperature, from the samples that have NOT been 
cryo-protected yet.

You need to figure out if the cryo-protection and/or cryo-cooling are damaging 
your crystals.

Best,

Nukri


Ruslan Sanishvili (Nukri), Ph.D.
Macromolecular Crystallographer
GM/CA@APS
X-ray Science Division, ANL
9700 S. Cass Ave.
Lemont, IL 60439

Tel: (630)252-0665
Fax: (630)252-0667
rsanishv...@anl.gov




From: CCP4 bulletin board  on behalf of Tang Chenjun 
<0910010...@cau.edu.cn>
Sent: Friday, July 28, 2017 2:21 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] weird diffraction pattern

Hi,

Thanks to all who gave me suggestions concerning the weird diffraction pattern 
and I really appreciate it that Kay Diederichs help me processing my data set 
and answer my questions. Although the data set can be processed using HKL3000, 
XDS without problems, the Rwork/Rfree values are still above 0.5 after 
molecular replacement. There can be several reasons.
1) The structure change a lot after binding DNA, so it is not possible to find 
a solution using molecular replacement.
2) Strong radiation damage and 1.0 degree image widths prevent good integration 
results. It may be better to use 0.1 degree image widths.
3) Streaky spots appearing in certain directions because of anisotropy or 
lattice translocation disorder, or one very large unit cell dimension lying 
along the X-ray beam may also have an affect on data processing.

Now I am optimizing the crystals to address these problems.

Best wishes and thanks again for your help,

Chenjun Tang

Re: [ccp4bb] weird diffraction pattern

[Tang Chenjun]

Hi,

Thanks to all who gave me suggestions concerning the weird diffraction pattern 
and I really appreciate it that Kay Diederichs help me processing my data set 
and answer my questions. Although the data set can be processed using HKL3000, 
XDS without problems, the Rwork/Rfree values are still above 0.5 after 
molecular replacement. There can be several reasons.
1) The structure change a lot after binding DNA, so it is not possible to find 
a solution using molecular replacement.
2) Strong radiation damage and 1.0 degree image widths prevent good integration 
results. It may be better to use 0.1 degree image widths.
3) Streaky spots appearing in certain directions because of anisotropy or 
lattice translocation disorder, or one very large unit cell dimension lying 
along the X-ray beam may also have an affect on data processing.

Now I am optimizing the crystals to address these problems. 

Best wishes and thanks again for your help,

Chenjun Tang