[ccp4bb] few questions about resolving new structure through MR

2013-11-07 Thread Zhihong Yu
Hi, all

I'm a rookie in resolving a brand new structure. I have some questions for
my current case and look forward to some suggestions.

Now I’m working on a protein like this:
N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a
diffraction data just to 3.5Å, and there is no complete homology structure
in pdb bank, but only a homology structure (named as structureX later) for
domainB with ~30% sequence identity, so I have some questions as following:

1. Is it possible to find a resolution through MR approach using structureX
as a search model? Especially considering that the resolution is only 3.5Å.
Currently I just tried once using phaser and refine the structure, I can
get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the
structureX, especially those within helix or sheet, can be well described
by 2Fo-Fc density. Is this primary result promising or not?

2. If it’s possible, what’s the general optimal procedure I should follow?

Really thanks for any advice and suggestions!

Zhihong


Re: [ccp4bb] few questions about resolving new structure through MR

2013-11-07 Thread Roger Rowlett
Count yourself lucky that you may have a partial solution for your 
structure with only 30% identity. The question now is: can you see any 
reasonable, traceable electron density for domain A?


Cheers,

___
Roger S. Rowlett
Gordon  Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 11/7/2013 11:36 AM, Zhihong Yu wrote:


Hi, all

I'm a rookie in resolving a brand new structure. I have some questions 
for my current case and look forward to some suggestions.


Now I’m working on a protein like this: 
N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I 
got a diffraction data just to 3.5Å, and there is no complete homology 
structure in pdb bank, but only a homology structure (named as 
structureX later) for domainB with ~30% sequence identity, so I have 
some questions as following:


1. Is it possible to find a resolution through MR approach using 
structureX as a search model? Especially considering that the 
resolution is only 3.5Å. Currently I just tried once using phaser and 
refine the structure, I can get a R/Rfree of 0.45/0.55, and it looks 
like most of backbone in the structureX, especially those within helix 
or sheet, can be well described by 2Fo-Fc density. Is this primary 
result promising or not?


2. If it’s possible, what’s the general optimal procedure I should 
follow?


Really thanks for any advice and suggestions!

Zhihong





Re: [ccp4bb] few questions about resolving new structure through MR

2013-11-07 Thread Greg Costakes
The fact that you have a 10% split between R/Rfree means your solution is 
heavily model biased (rule of thumb is a split of 5%). An Rfree of 0.55 would 
imply randomness. So unfortunately in this case, I dont think that you have an 
actual solution. You could try MR with a poly-A form of the homology model to 
see if you get a better phaser solution. Then proceed with the refinement while 
being careful to keep the R/Rfree within 5% and slowly build in the residues of 
the rest of your protein based on adequate electron density. Hope this helps. 

- Greg 

--- 
Greg Costakes, Ph.D. 
Department of Structural Biology 
Purdue University 
Hockmeyer Hall, Room 320 
240 S. Martin Jischke Drive, West Lafayette, IN 47907 


 


- Original Message -
From: Zhihong Yu nkyuz...@gmail.com 
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Thursday, November 7, 2013 11:36:51 AM 
Subject: [ccp4bb] few questions about resolving new structure through MR 




Hi, all 

I'm a rookie in resolving a brand new structure. I have some questions for my 
current case and look forward to some suggestions. 

Now I’m working on a protein like this: 
N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a 
diffraction data just to 3.5Å, and there is no complete homology structure in 
pdb bank, but only a homology structure (named as structureX later) for domainB 
with ~30% sequence identity, so I have some questions as following: 

1. Is it possible to find a resolution through MR approach using structureX as 
a search model? Especially considering that the resolution is only 3.5Å. 
Currently I just tried once using phaser and refine the structure, I can get a 
R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, 
especially those within helix or sheet, can be well described by 2Fo-Fc 
density. Is this primary result promising or not? 

2. If it’s possible, what’s the general optimal procedure I should follow? 

Really thanks for any advice and suggestions! 

Zhihong

Re: [ccp4bb] few questions about resolving new structure through MR

2013-11-07 Thread Zhihong Yu
First of all, thanks so much for your reply.
 To Roger:
NO, unfortunately I cannot see too much traceable electron density outside
the placed atoms, so I think just as Greg said, it's only a model-biased
solution.
 To Greg:
YES, I also realized that the input model should be very important, so I'm
going to try only backbone of structureX, or build a homology model of
domainB first and then put it as a search model. Actually, I asked those
questions because I had no idea that even I can correctly place domainB
using structureX as a search model, can I really resolve the full length
structure? after all, the resolution is only 3.5, and the domainB is only
contain 40% residues of the full length. I really want to get some opinions
from you expert whether it's worth to spend much time on trying to resolve
the strucutre through MR based on current dataset. Or I have to prepare
SeMet protein to get experimental phasing information?
Thank you all again and look forward to hearing from more expert!
Zhihong
On Thu, Nov 7, 2013 at 1:25 PM, Greg Costakes gcost...@purdue.edu wrote:

 The fact that you have a 10% split between R/Rfree means your solution is
 heavily model biased (rule of thumb is a split of 5%). An Rfree of 0.55
 would imply randomness. So unfortunately in this case, I dont think that
 you have an actual solution. You could try MR with a poly-A form of the
 homology model to see if you get a better phaser solution. Then proceed
 with the refinement while being careful to keep the R/Rfree within 5% and
 slowly build in the residues of the rest of your protein based on adequate
 electron density. Hope this helps.

  - Greg


 ---
 Greg Costakes, Ph.D.
 Department of Structural Biology
 Purdue University
 Hockmeyer Hall, Room 320
 240 S. Martin Jischke Drive, West Lafayette, IN 47907


 


 --
 *From: *Zhihong Yu nkyuz...@gmail.com
 *To: *CCP4BB@JISCMAIL.AC.UK
 *Sent: *Thursday, November 7, 2013 11:36:51 AM
 *Subject: *[ccp4bb] few questions about resolving new structure through MR


 Hi, all

 I'm a rookie in resolving a brand new structure. I have some questions for
 my current case and look forward to some suggestions.

 Now I’m working on a protein like this:
 N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a
 diffraction data just to 3.5Å, and there is no complete homology structure
 in pdb bank, but only a homology structure (named as structureX later) for
 domainB with ~30% sequence identity, so I have some questions as following:

 1. Is it possible to find a resolution through MR approach using
 structureX as a search model? Especially considering that the resolution is
 only 3.5Å. Currently I just tried once using phaser and refine the
 structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of
 backbone in the structureX, especially those within helix or sheet, can be
 well described by 2Fo-Fc density. Is this primary result promising or not?

 2. If it’s possible, what’s the general optimal procedure I should follow?

 Really thanks for any advice and suggestions!

 Zhihong



Re: [ccp4bb] few questions about resolving new structure through MR

2013-11-07 Thread Tim Gruene
-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Zhihong,

in many labs a SeMet prep is not much effort and can be done within a
week or two, if you express in E.coli. Unless the costs are a limiting
factor I would certainly go this way. However, with native data to
3.5A I suggest you contact somebody knowledgeable to help you collect
a MAD data set - this will be difficult enough.

While the protein is being expressed and purified you can start a
couple of MR jobs with different search models - I would use sculptor
or mrtailor, though, instead of a plain poly-ALA model: don't make
life more complicated than necessary.

Regards,
Tim

On 11/07/2013 08:31 PM, Zhihong Yu wrote:
 First of all, thanks so much for your reply. To Roger: NO,
 unfortunately I cannot see too much traceable electron density
 outside the placed atoms, so I think just as Greg said, it's only a
 model-biased solution. To Greg: YES, I also realized that the input
 model should be very important, so I'm going to try only backbone
 of structureX, or build a homology model of domainB first and then
 put it as a search model. Actually, I asked those questions because
 I had no idea that even I can correctly place domainB using
 structureX as a search model, can I really resolve the full length 
 structure? after all, the resolution is only 3.5, and the domainB
 is only contain 40% residues of the full length. I really want to
 get some opinions from you expert whether it's worth to spend much
 time on trying to resolve the strucutre through MR based on current
 dataset. Or I have to prepare SeMet protein to get experimental
 phasing information? Thank you all again and look forward to
 hearing from more expert! Zhihong On Thu, Nov 7, 2013 at 1:25 PM,
 Greg Costakes gcost...@purdue.edu wrote:
 
 The fact that you have a 10% split between R/Rfree means your
 solution is heavily model biased (rule of thumb is a split of
 5%). An Rfree of 0.55 would imply randomness. So unfortunately
 in this case, I dont think that you have an actual solution. You
 could try MR with a poly-A form of the homology model to see if
 you get a better phaser solution. Then proceed with the
 refinement while being careful to keep the R/Rfree within 5% and 
 slowly build in the residues of the rest of your protein based on
 adequate electron density. Hope this helps.
 
 - Greg
 
 
 ---

 
Greg Costakes, Ph.D.
 Department of Structural Biology Purdue University Hockmeyer
 Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN
 47907
 
 
 



 
- --
 *From: *Zhihong Yu nkyuz...@gmail.com *To:
 *CCP4BB@JISCMAIL.AC.UK *Sent: *Thursday, November 7, 2013
 11:36:51 AM *Subject: *[ccp4bb] few questions about resolving new
 structure through MR
 
 
 Hi, all
 
 I'm a rookie in resolving a brand new structure. I have some
 questions for my current case and look forward to some
 suggestions.
 
 Now I’m working on a protein like this: 
 N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa),
 I got a diffraction data just to 3.5Å, and there is no complete
 homology structure in pdb bank, but only a homology structure
 (named as structureX later) for domainB with ~30% sequence
 identity, so I have some questions as following:
 
 1. Is it possible to find a resolution through MR approach using 
 structureX as a search model? Especially considering that the
 resolution is only 3.5Å. Currently I just tried once using phaser
 and refine the structure, I can get a R/Rfree of 0.45/0.55, and
 it looks like most of backbone in the structureX, especially
 those within helix or sheet, can be well described by 2Fo-Fc
 density. Is this primary result promising or not?
 
 2. If it’s possible, what’s the general optimal procedure I
 should follow?
 
 Really thanks for any advice and suggestions!
 
 Zhihong
 
 

- -- 
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

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Re: [ccp4bb] few questions about resolving new structure through MR

2013-11-07 Thread Francis Reyes
Do you expect more than one molecule in the asymmetric unit?

Determined from the Matthews Coefficient (poor), size exclusion column 
(better), or self RF (best) ? 


On Nov 7, 2013, at 8:36 AM, Zhihong Yu nkyuz...@gmail.com wrote:

 Hi, all
 
 I'm a rookie in resolving a brand new structure. I have some questions for my 
 current case and look forward to some suggestions.
 
 Now I’m working on a protein like this: 
 N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a 
 diffraction data just to 3.5Å, and there is no complete homology structure in 
 pdb bank, but only a homology structure (named as structureX later) for 
 domainB with ~30% sequence identity, so I have some questions as following:
 
 1. Is it possible to find a resolution through MR approach using structureX 
 as a search model? Especially considering that the resolution is only 3.5Å. 
 Currently I just tried once using phaser and refine the structure, I can get 
 a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, 
 especially those within helix or sheet, can be well described by 2Fo-Fc 
 density. Is this primary result promising or not?
 
 2. If it’s possible, what’s the general optimal procedure I should follow?
 
 Really thanks for any advice and suggestions!
 
 Zhihong
 

-
Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder


Re: [ccp4bb] few questions about resolving new structure through MR

2013-11-07 Thread Zhihong Yu
Thanks Francis,

No, only one molecule in the asu. The Matthews Coefficient is 3.3,
corresponding solvent content is 62.6%, maybe that's why this crystal show
such weak diffraction?

Zhihong


On Thu, Nov 7, 2013 at 5:37 PM, Francis Reyes francis.re...@colorado.eduwrote:


 Do you expect more than one molecule in the asymmetric unit?

 Determined from the Matthews Coefficient (poor), size exclusion column
 (better), or self RF (best) ?


 On Nov 7, 2013, at 8:36 AM, Zhihong Yu nkyuz...@gmail.com wrote:

  Hi, all
 
  I'm a rookie in resolving a brand new structure. I have some questions
 for my current case and look forward to some suggestions.
 
  Now I’m working on a protein like this:
 N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a
 diffraction data just to 3.5Å, and there is no complete homology structure
 in pdb bank, but only a homology structure (named as structureX later) for
 domainB with ~30% sequence identity, so I have some questions as following:
 
  1. Is it possible to find a resolution through MR approach using
 structureX as a search model? Especially considering that the resolution is
 only 3.5Å. Currently I just tried once using phaser and refine the
 structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of
 backbone in the structureX, especially those within helix or sheet, can be
 well described by 2Fo-Fc density. Is this primary result promising or not?
 
  2. If it’s possible, what’s the general optimal procedure I should
 follow?
 
  Really thanks for any advice and suggestions!
 
  Zhihong
 

 -
 Francis E. Reyes PhD
 215 UCB
 University of Colorado at Boulder









Re: [ccp4bb] few questions about resolving new structure through MR

2013-11-07 Thread Zhihong Yu
Dear Debanu,

Thanks for your detailed reply. The Z-Score in my current MR trial is only
4.2, which means that domainB was not correctly placed at all, the observed
density is indeed model biased density. Since it's my first experience of
resolving a new structure, I'm really not sure whether it's worth to put
too much efforts on MR based on current 3.5A dataset and only a structure
with low homology with one domain. From your reply, I think it's still
worth to try a little bit and got information as much as I can. I'm going
to try MR Rosetta first.

Best Regards!

Zhihong


On Thu, Nov 7, 2013 at 6:36 PM, Das, Debanu deb...@slac.stanford.eduwrote:

 Hi Zhihong,

 The 3.5A diffraction could be due to many reasons: N- and C-term regions,
 interdomain linker possibly giving rise to molecular flexibility, quality
 of the particular crystals, cryo, purification, tags, etc.

 One thing to try is to run secondary structure predictions (or BLAST
 against PDB, FFAS) on the N- and C-term regions and optimize your construct
 to exclude some or all of them, especially if you have evidence that they
 might not be functionally important.

 1) Observing density corresponding to your protein sounds promising. What
 is your PHASER Z-score? Usually Z-scores  8 are indicative of correct
 solutions so if you are confident that you have the correct
 placement/solution for domain B, you can try to optimize refinement/model
 using DEN or MR Rosetta or morph_model.

 2) Try the above and see if you can improve your model/maps/R-values. Try
 optimizing your model (changing residues, removing loops, etc.) by homology
 modeling (you can try using the PSI Modeling Portal
 http://www.proteinmodelportal.org/) or other similar services or try
 different programs individually.

 In addition, try to obtain a homology model of domainA (including model
 building with Rosetta/Robetta).

 Additional phasing information by experimental phasing using SeMet or
 heavy atoms will be best, but is often easier said than done. Since you are
 at the MR stage, it will be useful if you can squeeze as much information
 as you can from MR efforts. If you are sure you have domainB placed
 correctly (and can also obtain a reliable solution for domainA), your MR
 phases can be used later on to locate heavy atom sites by difference
 Fourier methods and you can also combine with experimental phases in
 non-optimal cases

 Best,
 Debanu.
 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Zhihong Yu
 [nkyuz...@gmail.com]
 Sent: Thursday, November 07, 2013 2:53 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] few questions about resolving new structure through
 MR

 Thanks Francis,

 No, only one molecule in the asu. The Matthews Coefficient is 3.3,
 corresponding solvent content is 62.6%, maybe that's why this crystal show
 such weak diffraction?

 Zhihong


 On Thu, Nov 7, 2013 at 5:37 PM, Francis Reyes francis.re...@colorado.edu
 mailto:francis.re...@colorado.edu wrote:

 Do you expect more than one molecule in the asymmetric unit?

 Determined from the Matthews Coefficient (poor), size exclusion column
 (better), or self RF (best) ?


 On Nov 7, 2013, at 8:36 AM, Zhihong Yu nkyuz...@gmail.commailto:
 nkyuz...@gmail.com wrote:

  Hi, all
 
  I'm a rookie in resolving a brand new structure. I have some questions
 for my current case and look forward to some suggestions.
 
  Now I’m working on a protein like this:
 N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a
 diffraction data just to 3.5Å, and there is no complete homology structure
 in pdb bank, but only a homology structure (named as structureX later) for
 domainB with ~30% sequence identity, so I have some questions as following:
 
  1. Is it possible to find a resolution through MR approach using
 structureX as a search model? Especially considering that the resolution is
 only 3.5Å. Currently I just tried once using phaser and refine the
 structure, I can get a R/Rfree of 0.45/0.55, and it looks like most of
 backbone in the structureX, especially those within helix or sheet, can be
 well described by 2Fo-Fc density. Is this primary result promising or not?
 
  2. If it’s possible, what’s the general optimal procedure I should
 follow?
 
  Really thanks for any advice and suggestions!
 
  Zhihong
 

 -
 Francis E. Reyes PhD
 215 UCB
 University of Colorado at Boulder