[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 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 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









[ccp4bb] The density shown in Pymol and Coot is different.

2011-01-07 Thread Zhihong Yu
Hi,all

I refined a protein-ligand complex structure using Refmac5 and got the map
coefficient A.mtz file. I want to represent the electron density in Pymol,
so I transformed A.mtz into A.map ccp4-format map file using FFT in
ccp4i (parameters are: generate simple map in CCP4 format to cover
asymmetric unit, all others are default). When I load A.map inot Pymol
and show eletron density around the ligand, I found the electron density was
somewhat different comparing to that shown in coot using A.mtz, density in
Pymol (using A.map) was much stronger than that in Coot (using A.mtz)
when showing at 1.0 sigma level. Where does the differenc come from? Should
I set any other parameters when I transform mtz to map using FFT program?

Thanks in advance!

Zhihong


Re: [ccp4bb] The density shown in Pymol and Coot is different.

2011-01-07 Thread Zhihong Yu
Hi, Nian,

Thanks for your reply. I figured out the problem just now. The difference
indeed came from my FFT transformation. I just used all default parameters
when I transform A.mtz to A.map, so in the Run FFT dialog, F1= F,
Sigma=SIGF, PHI=PHIC, Weight=Unaasigned, and I got A.map under these
conditions. Even when I load A.map into Coot, the density is different
with A.mtz. But when I assigned parameters as following: F1= FWT,
Sigma=Unaasigned, PHI=PHWT, Weight=Unaasigned, and ran the job, I got
another A1.map, the density is exactly same as A.mtz this time whatever
in Pymol or Coot. I'm a rookie in this field, so herein I have another
question about this: Which map should I use for showing the final density,
A.map or A1.map? What's the difference of these two maps, it looks like
A.map is much stronger than A1.map.

Really thanks for response!

Zhihong



2011/1/7 Nian Huang huangn...@gmail.com

 It has been discussed before. Nothing to do with FFT. Both pymol and
 coot will rescale your map but differently. You have to really look
 into how the programmer wrote their program to know what is going on.
 For now, I suggest you just use coot map which is more realistic to
 me. Also new version of pymol seems to work better.

 On Fri, Jan 7, 2011 at 1:42 PM, Zhihong Yu nkyuz...@gmail.com wrote:
  Hi,all
 
  I refined a protein-ligand complex structure using Refmac5 and got the
 map
  coefficient A.mtz file. I want to represent the electron density in
 Pymol,
  so I transformed A.mtz into A.map ccp4-format map file using FFT in
  ccp4i (parameters are: generate simple map in CCP4 format to cover
  asymmetric unit, all others are default). When I load A.map inot
 Pymol
  and show eletron density around the ligand, I found the electron density
 was
  somewhat different comparing to that shown in coot using A.mtz, density
 in
  Pymol (using A.map) was much stronger than that in Coot (using A.mtz)
  when showing at 1.0 sigma level. Where does the differenc come from?
 Should
  I set any other parameters when I transform mtz to map using FFT program?
 
  Thanks in advance!
 
  Zhihong



Re: [ccp4bb] The density shown in Pymol and Coot is different.

2011-01-07 Thread Zhihong Yu
Matthew  Nat,

Thanks very much, your answers made me much clear about the map
manipulating, as well as usage of Refmac and FFT. Just from my results, I
think I agree with Nat that Sigma and Weight is unnecessary when running
FFT based on a Refmac-resulted mtz file, since my A1.map is exactly same
as FWT of A.mtz in Coot.

Again thanks for your help!

Zhihong

2011/1/7 Nat Echols nathaniel.ech...@gmail.com

 On Fri, Jan 7, 2011 at 2:34 PM, Matthew Franklin mfrank...@nysbc.orgwrote:

  However, I'm not sure if you're doing the right thing by leaving SIGF
 unassigned.  I believe this should be assigned to the experimental error,
 which is usually called 'SIGF' in your mtz file.  I don't know if FFT
 actually uses this for anything when it's calculating your map file.  I'm
 also not sure what you should use for the weight parameter - perhaps 'FOM'?


 FWT,PHWT has the weights already applied to the amplitudes*, so no FOM is
 necessary.  According to the FFT documentation, the sigmas can be used to
 exclude reflections (subject to an additional keyword), but I assume by
 default it will just be ignored.

 -Nat

 (* FWT = 2mFo-DFc, where m is the FOM.)