Re: [ccp4bb] Big difference between anomalous map and density modification map
Hi, Clemens,
sorry for the confusing and unclear expression of my case.
>Confused ... what program/procedure did you use for what you call "density
>modification" and what for "solvent flattening"?
my solvent content is supposed to be 65% (one molecule per ASU). Sharp
automatically did density modification using 37.5% solvent content. that is why
I run solvent flattening directly from Sharp.
>More confusion: what do you mean with "anomalous map"? One might call a map
>with anomalous differences and some phases such an "anomalous map" ... but I
>guess you rather mean an electron density map with phases from some procedure
>based on anomalous differences, right?
you are right. the anomalous map I calculated is anomalous differences map
using FFT
> fft hklin eden_flat_53.2pc.mtz mapout eden_flat_53.2pc.map < LABI F1=FBshasol PHI=PHIBshasol
> fft hklin eden_flat_53.2pc.mtz mapout eden_flat_53.2pc.map < LABI F1=FBshasol PHI=PHIBshasol e
yes, I calculated both density maps. the density around all 6 selenium sites is
poor.
Thank you very much.
mousheng
Mousheng Wu, PhD
Center for Membrane Biology
Department of Biochemistry and Molecular Biology
The University of Texas Medical School at Houston
6431 Fannin Street, Houston, TX, USA, 77030
From: Clemens Vonrhein [vonrh...@globalphasing.com]
Sent: Monday, October 18, 2010 8:05 AM
To: Wu, Mousheng
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Big difference between anomalous map and density
modification map
Dear Mousheng,
as others, I'm also a bit confused about the steps you did:
On Sun, Oct 17, 2010 at 02:29:12PM -0500, Wu, Mousheng wrote:
> Dear All,
>
> I have a MAD dataset at 4Å and shelxD can find clear-cut 10
> solutions (my protein has 12 methionines).
Good.
> I ran autosharp to refine them followed by density modification.
Yes.
> After density modification, I ran solvent flattening.
Confused ... what program/procedure did you use for what you call
"density modification" and what for "solvent flattening"?
> Then I calculated the anomalous map using the phase from sharp and
> the electron density map from solvent flattening.
More confusion: what do you mean with "anomalous map"? One might call
a map with anomalous differences and some phases such an "anomalous
map" ... but I guess you rather mean an electron density map with
phases from some procedure based on anomalous differences, right?
So you calculated an electron density map from SHARP: which map
coefficients did you use? It should be:
fft hklin eden.mtz mapout eden.map < The anomalous map shows the density around these 10 selenium sites
> are clear and round.
Good.
> However, the density map from solvent flattening showed that only 4
> selenium sites have clear and round density.
Is the map after "density modifcation" or the one after "density
modification plus solvent flattening"? It is very unclear ...
> The density around these 4 sites clearly showed beautiful
> helices. surprisingly, other 6 selenium sites have poor density or
> no density at all. The electron density around them is not very
> good and the predicted helical density is flat. I check the electron
> density before I ran solvent flattening. The result is same. I am
> quite confused about the big difference from these two maps.
We first need to establish what steps you performed and how to see if
these are the actually correct maps to look at.
> I also try SnB to find the selenium sites. The solutions are same as
> those from ShelxD. But How to explain the poor density around the
> selenium sites in the density modification map? Is there any
> problem with my selenium sites?
Remember that the purely HA-phased maps (i.e. directly after SHARP)
will be dominated by those heavy atoms ... so no surprise they are
nice and round Se density.
Se-MET (or S-Met for that matter) often has alternate conformations in
the side-chain: maybe your phases are actually better and therefore
model those side-chains now more accurately, i.e. as disordered
side-chains. If your environment around those sites is also disorderd
('flattened' helices) this is all not surprising.
Cheers
Clemens
--
***
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
*
* Global Phasing Ltd.
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[ccp4bb] Big difference between anomalous map and density modification map
Dear All, I have a MAD dataset at 4Å and shelxD can find clear-cut 10 solutions (my protein has 12 methionines). I ran autosharp to refine them followed by density modification. After density modification, I ran solvent flattening. Then I calculated the anomalous map using the phase from sharp and the electron density map from solvent flattening. The anomalous map shows the density around these 10 selenium sites are clear and round. However, the density map from solvent flattening showed that only 4 selenium sites have clear and round density.The density around these 4 sites clearly showed beautiful helices. surprisingly, other 6 selenium sites have poor density or no density at all. The electron density around them is not very good and the predicted helical density is flat. I check the electron density before I ran solvent flattening. The result is same. I am quite confused about the big difference from these two maps. I also try SnB to find the selenium sites. The solutions are same as those from ShelxD. But How to explain the poor density around the selenium sites in the density modification map? Is there any problem with my selenium sites? Any suggestion from crystallographic experts? Thanks. Mousheng Wu, PhD Center for Membrane Biology Department of Biochemistry and Molecular Biology The University of Texas Medical School at Houston 6431 Fannin Street, Houston, TX, USA, 77030
[ccp4bb] domains missing
Hi, ccp4 community, I am solving my protein (300 aa) structure using molecular replacement. The space group is P622. There is only one molecule in the ASU. The protein is supported to have three domains. We have solved the domain 1 (120aa) structure; therefore we tried to use it as a model to solve the new structure. MolRep and Phaser can find the domain 1. We refined the model and the R and Rfree factor was around 50%. When we used Coot to see the model and map, we found that the other two domains are missing. In the map, there is a big 'hole' and little extra density (with 6-fold) inside. It is a big surprise to us. Is there any possibility that R is around 50 if the other two domains are missing? Any suggests to figure out what happen to my structure? Thanks a lot! Mousheng Wu
[ccp4bb] struggling with molecular replacement
hi, everyone! I am struggling with my crystal structure in a very big unit cell. my protein is about 16KDa. probably there are about 20 molecules in the asymmetric unit. I tried to use molecular replacement to solve the structure. However, there is only one NMR structure available for me. The second trouble is there are so many molecules in the ASU. I tried use NMR structure as a model to solve it. MolRep gave me 4 solutions. I refined it first and see the map. The map looks quite reasonable. some parts of the model fit the density and some do not . i tried to trace one molecule first. you can image how hard it is. Does anyone have such experience? how can i find so many solutions with NMR model? your suggestions are much appreciated! Mousheng Wu
[ccp4bb] oxalate
As Anthony and Jeremy suggested, I tried to fit oxalate into the density. It perfectly matches the density. Thank all of you. I should read more papers now to make it sense.
Re: [ccp4bb] unknown density in structure
There are four molecules in the asymmetric unit. And this density is present in every molecules. -Original Message- From: ANA MARIA MISIC [mailto:[EMAIL PROTECTED] Sent: Thursday, July 26, 2007 5:24 PM To: Wu, Mousheng Subject: Re: [ccp4bb] unknown density in structure Hi Mousheng - Did you check to see if that falls on a special position? That can lead to a buildup of electron density, and the symmetrical shape is suspect. good luck Ana M. Misic Research Assistant Department of Biomolecular Chemistry Department of Bacteriology University of Wisconsin - Madison 420 Henry Mall, Rm 230 Madison, WI 53706 Lab Phone: (608)265-9282 Fax: (608)262-9865 - Original Message - From: "Wu, Mousheng" <[EMAIL PROTECTED]> Date: Thursday, July 26, 2007 4:21 pm Subject: [ccp4bb] unknown density in structure To: CCP4BB@JISCMAIL.AC.UK > Hi, everyone, > > > > I am recently solving a structure at 1.8A. After I fitted all residues > into the model, I found that there was an extra density in a pocket > consisted of E, N, R and other hydrophobic residues. The density is > strong and connected like a butterfly. I checked the crystallization > condition. I can not figure out what it is. It seems that it is not from > the crystallization components. > > > > Does anyone know how to figure out what the density is? Is there any > program or database I can use to predict the potential ligand? > > Thanks. > > > > Mousheng >
[ccp4bb] unknown density in structure
Hi, everyone, I am recently solving a structure at 1.8A. After I fitted all residues into the model, I found that there was an extra density in a pocket consisted of E, N, R and other hydrophobic residues. The density is strong and connected like a butterfly. I checked the crystallization condition. I can not figure out what it is. It seems that it is not from the crystallization components. Does anyone know how to figure out what the density is? Is there any program or database I can use to predict the potential ligand? Thanks. Mousheng
