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Dear all:

I am trying to solve a structure from apparently a hexagonal crystal.  
I indexed and scaled data  in P6 in Scalepack (with merging) then used  
Scalepack2mtz (with ensure unique reflections and add Rfree as well as  
the truncate procedure), and then attempted to run molecular  
replacement with Phaser. Now the problem appeared - Phaser immediately  
quits with the following error message "FATAL RUNTIME ERROR:  
Reflections are not a unique set by symmetry". I do not understand  
this at all.

I also tried running scalepack using the NO MERGE macro as people have  
indicated earlier on this bb (thank you again!, I also checked the  
scl.in that is written out and it had the NO MERGE statement), and  
then tried to run pointless to verify the spacegroup but the program  
complained the reflections are merged (that is impossible, I checked  
the number of reflections in the unmerged and merged files and they  
were different as one would expect). I repeated the procedures several  
times and I always get the same errors. I can't make any sense of this  
and I can't move forward. Any ideas?

Many thanks,
Alex






On Jul 30, 2009, at 7:03 PM, CCP4BB automatic digest system wrote:

> There are 13 messages totalling 1026 lines in this issue.
>
> Topics of the day:
>
>  1. refmac failed message (2)
>  2. question of extra high B factor (6)
>  3. Coot:findwaters in REFMAC (2)
>  4. Postdoctoral position in protein crystallography at Karolinska  
> Institutet
>  5. OpenGL Stereo 3D on 120 Hz LCDs, at last!
>  6. Foils for energy calibration
>
> ----------------------------------------------------------------------
>
> Date:    Thu, 30 Jul 2009 10:50:13 GMT
> From:    Elad Binshtien <[log in to unmask]>
> Subject: refmac failed message
>
> This is a multi-part message in MIME format.
>
>
> ----8d5c55134e491a1d1bdd
> Content-Type: text/plain; charset=utf-8
> Content-Disposition: inline
> Content-Transfer-Encoding: quoted-printable
>
> Dear all=2C
>
>
> I am refining a structure in Refmac at 2=2E2 A in win OS=2E=C2=A0  
> Howeve=
> r=2C
> Refmac failed and send this message=3A =C2=A0 =C2=A0 =C2=A0=C2=A0  
> forrtl=
> =3A error (72)=3A floating overflow=C2=A0 =
>
>
> Thank you in advance for your any helpful suggestions=2E=C2=A0 =
>
>
>
> Best=2C
> Elad
>
>
> Elad Binshtein
> Ph=2ED student =
>
> Department of Life Science =
>
> Ben Gurion University of the Negev
> Ph=3A 972-8-6461325=E2=80=8E
>
> ----8d5c55134e491a1d1bdd
> Content-Type: text/html; charset=utf-8
> Content-Disposition: inline
> Content-Transfer-Encoding: quoted-printable
>
> Dear all=2C=3Cbr=3E=3Cbr=3E=3Cbr=3EI am refining a structure in  
> Refmac a=
> t 2=2E2 A in win OS=2E=26nbsp=3B However=2C
> Refmac failed and send this message=3A =26nbsp=3B =26nbsp=3B  
> =26nbsp=3B=26=
> nbsp=3B forrtl=3A error (72)=3A floating overflow=26nbsp=3B  
> =3Cbr=3E=3Cb=
> r=3E Thank you in advance for your any helpful  
> suggestions=2E=26nbsp=3B =
> = 
> 3Cbr 
> =3E=3Cbr=3E=3Cbr=3EBest=2C=3Cbr=3EElad=3Cbr=3E=3CBR=3E=3CBR=3EElad =
> Binshtein=3Cbr=3EPh=2ED student =3Cbr=3EDepartment of Life Science  
> =3Cbr=
> =3EBen Gurion University of the Negev=3Cbr=3EPh=3A 972-8-6461325=3C/ 
> BR=3E=
> =3C/BR=3E=E2=80=8E
>
> ----8d5c55134e491a1d1bdd--
>
> ------------------------------
>
> Date:    Thu, 30 Jul 2009 13:25:43 +0100
> From:    "Stein, ND (Norman)" <[log in to unmask]>
> Subject: Re: refmac failed message
>
> This is a multi-part message in MIME format.
>
> ------_=_NextPart_001_01CA1110.DB7CC0CB
> Content-Type: text/plain;
> 	charset="windows-1255"
> Content-Transfer-Encoding: quoted-printable
>
> Hi Elad
> =20
> You don't say which version of Refmac you are using but I think the =
> first thing to do would be to try the latest version (5.5.0102). You  
> can =
> pick this up from
> =20
> ftp://ftp.ccp4.ac.uk/nds/windows/refmac_5.5.0102/refmac5.exe
> =20
> Copy this exe file to the bin subdirectory of your CCP4  
> installation. =
> (Probably wise to make a backup copy of the existing refmac5.exe  
> first).
> =20
> Best wishes
> =20
> Norman Stein
> CCP4
>
> ________________________________
>
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf  
> Of =
> Elad Binshtien
> Sent: 30 July 2009 11:50
> To: [log in to unmask]
> Subject: [ccp4bb] refmac failed message
>
>
> Dear all,
>
>
> I am refining a structure in Refmac at 2.2 A in win OS.  However,  
> Refmac =
> failed and send this message:        forrtl: error (72): floating =
> overflow =20
>
> Thank you in advance for your any helpful suggestions. =20
>
>
> Best,
> Elad
>
>
> Elad Binshtein
> Ph.D student=20
> Department of Life Science=20
> Ben Gurion University of the Negev
> Ph: 972-8-6461325
>
> =FD=20
>
> -- =0AScanned by iCritical.=0A
>
> ------_=_NextPart_001_01CA1110.DB7CC0CB
> Content-Type: text/html;
> 	charset="windows-1255"
> Content-Transfer-Encoding: quoted-printable
>
> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0 Transitional//EN">
> <HTML><HEAD>
> <META http-equiv=3DContent-Type content=3D"text/html; =
> charset=3Dwindows-1255">
> <META content=3D"MSHTML 6.00.2900.3562" name=3DGENERATOR></HEAD>
> <BODY>
> <DIV dir=3Dltr align=3Dleft><SPAN class=3D419481712-30072009><FONT =
> face=3DArial=20
> color=3D#0000ff size=3D2>Hi Elad</FONT></SPAN></DIV>
> <DIV dir=3Dltr align=3Dleft><SPAN class=3D419481712-30072009><FONT =
> face=3DArial=20
> color=3D#0000ff size=3D2></FONT></SPAN>&nbsp;</DIV>
> <DIV dir=3Dltr align=3Dleft><SPAN class=3D419481712-30072009><FONT =
> face=3DArial=20
> color=3D#0000ff size=3D2>You don't say which version of Refmac you  
> are =
> using but I=20
> think the first thing to do would be to try the latest =
> version&nbsp;(5.5.0102).=20
> You can pick this up from</FONT></SPAN></DIV>
> <DIV dir=3Dltr align=3Dleft><SPAN class=3D419481712-30072009><FONT =
> face=3DArial=20
> color=3D#0000ff size=3D2></FONT></SPAN>&nbsp;</DIV>
> <DIV dir=3Dltr align=3Dleft><SPAN class=3D419481712-30072009><FONT =
> face=3DArial=20
> color=3D#0000ff size=3D2><A=20
> href=3D"ftp://ftp.ccp4.ac.uk/nds/windows/refmac_5.5.0102/ 
> refmac5.exe">ftp=
> ://ftp.ccp4.ac.uk/nds/windows/refmac_5.5.0102/refmac5.exe</A></ 
> FONT></SPA=
> N></DIV>
> <DIV dir=3Dltr align=3Dleft><SPAN class=3D419481712-30072009><FONT =
> face=3DArial=20
> color=3D#0000ff size=3D2></FONT></SPAN>&nbsp;</DIV>
> <DIV dir=3Dltr align=3Dleft><SPAN class=3D419481712-30072009><FONT =
> face=3DArial=20
> color=3D#0000ff size=3D2>Copy this exe file to the bin subdirectory  
> of=20
> your&nbsp;CCP4 installation. (Probably wise&nbsp;to make a backup  
> copy =
> of the=20
> existing refmac5.exe first).</FONT></SPAN></DIV>
> <DIV dir=3Dltr align=3Dleft><SPAN class=3D419481712-30072009><FONT =
> face=3DArial=20
> color=3D#0000ff size=3D2></FONT></SPAN>&nbsp;</DIV>
> <DIV dir=3Dltr align=3Dleft><SPAN class=3D419481712-30072009><FONT =
> face=3DArial=20
> color=3D#0000ff size=3D2>Best wishes</FONT></SPAN></DIV>
> <DIV dir=3Dltr align=3Dleft><SPAN class=3D419481712-30072009><FONT =
> face=3DArial=20
> color=3D#0000ff size=3D2></FONT></SPAN>&nbsp;</DIV>
> <DIV dir=3Dltr align=3Dleft><SPAN class=3D419481712-30072009><FONT =
> face=3DArial=20
> color=3D#0000ff size=3D2>Norman Stein</FONT></SPAN></DIV>
> <DIV dir=3Dltr align=3Dleft><SPAN class=3D419481712-30072009><FONT =
> face=3DArial=20
> color=3D#0000ff size=3D2>CCP4</FONT></SPAN></DIV><BR>
> <DIV class=3DOutlookMessageHeader lang=3Den-us dir=3Dltr align=3Dleft>
> <HR tabIndex=3D-1>
> <FONT face=3DTahoma size=3D2><B>From:</B> CCP4 bulletin board=20
> [mailto:[log in to unmask]] <B>On Behalf Of </B>Elad=20
> Binshtien<BR><B>Sent:</B> 30 July 2009 11:50<BR><B>To:</B>=20
> [log in to unmask]<BR><B>Subject:</B> [ccp4bb] refmac failed=20
> message<BR></FONT><BR></DIV>
> <DIV></DIV>Dear all,<BR><BR><BR>I am refining a structure in Refmac  
> at =
> 2.2 A in=20
> win OS.&nbsp; However, Refmac failed and send this message: &nbsp; =
> &nbsp;=20
> &nbsp;&nbsp; forrtl: error (72): floating overflow&nbsp;  
> <BR><BR>Thank =
> you in=20
> advance for your any helpful suggestions.&nbsp;=20
> <BR><BR><BR>Best,<BR>Elad<BR><BR><BR>Elad Binshtein<BR>Ph.D student=20
> <BR>Department of Life Science <BR>Ben Gurion University of the =
> Negev<BR>Ph:=20
> 972-8-6461325<BR><BR>=FD
> <br>=
> <p>-- =0A<BR>Scanned by iCritical.=0A</p>
> <br>=
> </BODY></HTML>
>
> ------_=_NextPart_001_01CA1110.DB7CC0CB--
>
> ------------------------------
>
> Date:    Fri, 31 Jul 2009 00:00:25 +0800
> From:    Jiamu Du <[log in to unmask]>
> Subject: question of extra high B factor
>
> --00221504874fd6c69c046fee6655
> Content-Type: text/plain; charset=ISO-8859-1
> Content-Transfer-Encoding: 7bit
>
> Dear All,
> I am refining a structure of a complex between of 50kD protein and a  
> 20kD
> glycosylated protien. The data is of 2.9 A resolution. The wilson B  
> factor
> is as high as 86.3 A^2.
> The refinement seems well with R/Rf of 0.21/0.25. But the B-factor  
> is extra
> high. For the 50kD part, the average B factor is 76.5 A^2. But the B  
> factor
> of the 20 kD glycosylated protein is as high as 133.3 A^2. Although  
> the
> electron density looks fine, even the sugar chain is seen clearly.
> My question is:
> 1. How to reduce the B factor to a reasonable level?
> 2. If it can not be redueced, when I published it, is this value  
> acceptable?
> 3. In the same of similar resolutionIs, is there some other  
> structures like
> this situation?  A component or a subunit of the protein has a extra  
> high B
> factor as high as 130.
>
> Thanks and best wishes.
>
>
> -- 
> Jiamu Du, Ph.D.
> State Key Laboratory of Molecular Biology
> Institute of Biochemistry and Cell Biology
> Shanghai Institutes for Biological Sciences
> Chinese Academy of Sciences
> 320 Yue-Yang Road
> Shanghai 200031
> P. R. China
> Tel: +86-21-5492-1117
> E-mail: [log in to unmask]
>
> --00221504874fd6c69c046fee6655
> Content-Type: text/html; charset=ISO-8859-1
> Content-Transfer-Encoding: quoted-printable
>
> Dear All,<br>I am refining a structure of a complex between of 50kD  
> protein=
> and a 20kD glycosylated protien. The data is of 2.9 A resolution.  
> The wils=
> on B factor is as high as 86.3 A^2.<br>The refinement seems well  
> with R/Rf =
> of 0.21/0.25. But the B-factor is extra high. For the 50kD part, the  
> averag=
> e B factor is 76.5 A^2. But the B factor of the 20 kD glycosylated  
> protein =
> is as high as 133.3 A^2. Although the electron density looks fine,  
> even the=
> sugar chain is seen clearly.<br>
> My question is:<br>1. How to reduce the B factor to a reasonable  
> level?<br>=
> 2. If it can not be redueced, when I published it, is this value  
> acceptable=
> ?<br>3. In the same of similar resolutionIs, is there some other  
> structures=
> like this situation?=A0 A component or a subunit of the protein has  
> a extr=
> a high B factor as high as 130.<br>
> <br>Thanks and best wishes.<br><br clear=3D"all"><br>-- <br>Jiamu  
> Du, Ph.D.=
> <br>State Key Laboratory of Molecular Biology<br>Institute of  
> Biochemistry =
> and Cell Biology<br>Shanghai Institutes for Biological  
> Sciences<br>Chinese =
> Academy of Sciences<br>
> 320 Yue-Yang Road<br>Shanghai 200031<br>P. R. China<br>Tel:  
> +86-21-5492-111=
> 7<br>E-mail: <a href=3D"mailto:[log in to unmask]">[log in to unmask]</ 
> a><br>
>
> --00221504874fd6c69c046fee6655--
>
> ------------------------------
>
> Date:    Thu, 30 Jul 2009 09:32:26 -0700
> From:    Pavel Afonine <[log in to unmask]>
> Subject: Re: question of extra high B factor
>
> Hi Jiamu,
>
>> My question is:
>> 1. How to reduce the B factor to a reasonable level?
>> 3. In the same of similar resolutionIs, is there some other  
>> structures
>> like this situation?
>
> POLYGON tool is (one of) your friend(s) to answer this question (apart
> from debatable one about "a reasonable level"):
>
> Acta Cryst. D65, 297-300 (2009).
>
> Now it is available in PHENIX (from the latest nightly builds):
> http://www.phenix-online.org/
>
> Please let me know if you have any questions about it.
>
> Pavel.
>
> ------------------------------
>
> Date:    Thu, 30 Jul 2009 17:47:55 +0100
> From:    Clemens Vonrhein <[log in to unmask]>
> Subject: Re: question of extra high B factor
>
> Dear Jiamu,
>
> On Fri, Jul 31, 2009 at 12:00:25AM +0800, Jiamu Du wrote:
>> I am refining a structure of a complex between of 50kD protein and  
>> a 20kD
>> glycosylated protien. The data is of 2.9 A resolution. The wilson B  
>> factor
>> is as high as 86.3 A^2.
>> The refinement seems well with R/Rf of 0.21/0.25. But the B-factor  
>> is extra
>> high. For the 50kD part, the average B factor is 76.5 A^2. But the  
>> B factor
>> of the 20 kD glycosylated protein is as high as 133.3 A^2. Although  
>> the
>> electron density looks fine, even the sugar chain is seen clearly.
>> My question is:
>> 1. How to reduce the B factor to a reasonable level?
>
> How do you define 'reasonable'? And why would you want to reduce this
> anyway?
>
>  ( 50*76.5 + 20*133.3 ) / 70 = 92.7
>
> which seems fairly close to the Wilson B of 86.3, right?
>
>> 2. If it can not be redueced, when I published it, is this value  
>> acceptable?
>
> Better question: is it the right value? Remember it is
>
> results -> publish
>
> and not
>
>  publish <- results
>
> ;-)
>
> If they're correct than they are acceptable (I would accept those
> values).
>
>> 3. In the same of similar resolutionIs, is there some other  
>> structures like
>> this situation?  A component or a subunit of the protein has a  
>> extra high B
>> factor as high as 130.
>
> I'm sure hundreds of them ... but I'm sure you want your structure to
> stand on its own, so don't look too close at other structures and
> repeat the various mistakes we've all made and that are now set in
> stone in some old PDB file.
>
> Cheers
>
> Clemens
>
> -- 
>
> ***************************************************************
> * Clemens Vonrhein, Ph.D.     vonrhein AT GlobalPhasing DOT com
> *
> *  Global Phasing Ltd.
> *  Sheraton House, Castle Park
> *  Cambridge CB3 0AX, UK
> *--------------------------------------------------------------
> * BUSTER Development Group      (http://www.globalphasing.com)
> ***************************************************************
>
> ------------------------------
>
> Date:    Fri, 31 Jul 2009 00:02:42 +0800
> From:    Jiamu Du <[log in to unmask]>
> Subject: Re: question of extra high B factor
>
> --0022152d604d09ee86046fee6f07
> Content-Type: text/plain; charset=ISO-8859-1
> Content-Transfer-Encoding: 7bit
>
> I am using TLS refinement with Phenix for the structure refinement.
>
>
> On Fri, Jul 31, 2009 at 12:00 AM, Jiamu Du <[log in to unmask]> wrote:
>
>> Dear All,
>> I am refining a structure of a complex between of 50kD protein and  
>> a 20kD
>> glycosylated protien. The data is of 2.9 A resolution. The wilson B  
>> factor
>> is as high as 86.3 A^2.
>> The refinement seems well with R/Rf of 0.21/0.25. But the B-factor  
>> is extra
>> high. For the 50kD part, the average B factor is 76.5 A^2. But the  
>> B factor
>> of the 20 kD glycosylated protein is as high as 133.3 A^2. Although  
>> the
>> electron density looks fine, even the sugar chain is seen clearly.
>> My question is:
>> 1. How to reduce the B factor to a reasonable level?
>> 2. If it can not be redueced, when I published it, is this value
>> acceptable?
>> 3. In the same of similar resolutionIs, is there some other  
>> structures like
>> this situation?  A component or a subunit of the protein has a  
>> extra high B
>> factor as high as 130.
>>
>> Thanks and best wishes.
>>
>>
>> --
>> Jiamu Du, Ph.D.
>> State Key Laboratory of Molecular Biology
>> Institute of Biochemistry and Cell Biology
>> Shanghai Institutes for Biological Sciences
>> Chinese Academy of Sciences
>> 320 Yue-Yang Road
>> Shanghai 200031
>> P. R. China
>> Tel: +86-21-5492-1117
>> E-mail: [log in to unmask]
>>
>
>
>
> -- 
> Jiamu Du, Ph.D.
> State Key Laboratory of Molecular Biology
> Institute of Biochemistry and Cell Biology
> Shanghai Institutes for Biological Sciences
> Chinese Academy of Sciences
> 320 Yue-Yang Road
> Shanghai 200031
> P. R. China
> Tel: +86-21-5492-1117
> E-mail: [log in to unmask]
>
> --0022152d604d09ee86046fee6f07
> Content-Type: text/html; charset=ISO-8859-1
> Content-Transfer-Encoding: quoted-printable
>
> I am using TLS refinement with Phenix for the structure  
> refinement.<br><br>=
> <br><div class=3D"gmail_quote">On Fri, Jul 31, 2009 at 12:00 AM,  
> Jiamu Du <=
> span dir=3D"ltr">&lt;<a href=3D"mailto:[log in to unmask]">[log in to unmask] 
> =
> </a>&gt;</span> wrote:<br>
> <blockquote class=3D"gmail_quote" style=3D"border-left: 1px solid  
> rgb(204, =
> 204, 204); margin: 0pt 0pt 0pt 0.8ex; padding-left: 1ex;">Dear  
> All,<br>I am=
> refining a structure of a complex between of 50kD protein and a 20kD  
> glyco=
> sylated protien. The data is of 2.9 A resolution. The wilson B  
> factor is as=
> high as 86.3 A^2.<br>
> The refinement seems well with R/Rf of 0.21/0.25. But the B-factor  
> is extra=
> high. For the 50kD part, the average B factor is 76.5 A^2. But the B  
> facto=
> r of the 20 kD glycosylated protein is as high as 133.3 A^2.  
> Although the e=
> lectron density looks fine, even the sugar chain is seen clearly.<br>
>
> My question is:<br>1. How to reduce the B factor to a reasonable  
> level?<br>=
> 2. If it can not be redueced, when I published it, is this value  
> acceptable=
> ?<br>3. In the same of similar resolutionIs, is there some other  
> structures=
> like this situation?=A0 A component or a subunit of the protein has  
> a extr=
> a high B factor as high as 130.<br>
>
> <br>Thanks and best wishes.<br><br clear=3D"all"><br>-- <br>Jiamu  
> Du, Ph.D.=
> <br>State Key Laboratory of Molecular Biology<br>Institute of  
> Biochemistry =
> and Cell Biology<br>Shanghai Institutes for Biological  
> Sciences<br>Chinese =
> Academy of Sciences<br>
>
> 320 Yue-Yang Road<br>Shanghai 200031<br>P. R. China<br>Tel:  
> +86-21-5492-111=
> 7<br>E-mail: <a href=3D"mailto:[log in to unmask]"  
> target=3D"_blank">jiamudu=
> @[log in to unmask]</a><br>
> </blockquote></div><br><br clear=3D"all"><br>-- <br>Jiamu Du,  
> Ph.D.<br>Stat=
> e Key Laboratory of Molecular Biology<br>Institute of Biochemistry  
> and Cell=
> Biology<br>Shanghai Institutes for Biological Sciences<br>Chinese  
> Academy =
> of Sciences<br>
> 320 Yue-Yang Road<br>Shanghai 200031<br>P. R. China<br>Tel:  
> +86-21-5492-111=
> 7<br>E-mail: <a href=3D"mailto:[log in to unmask]">[log in to unmask]</ 
> a><br>
>
> --0022152d604d09ee86046fee6f07--
>
> ------------------------------
>
> Date:    Thu, 30 Jul 2009 10:24:25 -0700
> From:    Huiying Li <[log in to unmask]>
> Subject: Coot:findwaters in REFMAC
>
> I used Coot:findwaters facility in REFMAC (CCP4 6.1.1) to add waters  
> to
> the refined structure. Often for the well ordered water sites the
> routine added two water molecules in single water site, with their
> distance (~1 Angs) way shorter than allowed hydrogen bonding  
> distance. I
> have to remove the extra water molecules manually.
>
> Is this a intended feature? It seems to only create extra work to the
> user. What is the purpose of having different chain IDs for waters  
> output
> from this routine?
>
>
> Huiying Li, Ph. D
> Department of Molecular Biology and Biochemistry
> Natural Sciences I, Rm 2443
> University of California at Irvine
> Irvine, CA 92697, USA
> Tel: 949-824-4322(or -1953);  Fax: 949-824-3280
> email: [log in to unmask]
>
> ------------------------------
>
> Date:    Thu, 30 Jul 2009 18:33:01 +0100
> From:    Paul Emsley <[log in to unmask]>
> Subject: Re: Coot:findwaters in REFMAC
>
> Huiying Li wrote:
>> I used Coot:findwaters facility in REFMAC (CCP4 6.1.1) to add waters
>> to the refined structure. Often for the well ordered water sites the
>> routine added two water molecules in single water site, with their
>> distance (~1 Angs) way shorter than allowed hydrogen bonding  
>> distance.
>> I have to remove the extra water molecules manually.
>
> Sounds like an old version.  Things have improved.
>
>>
>> Is this a intended feature? It seems to only create extra work to the
>> user.
>
> :-(
>
>> What is the purpose of having different chain IDs for waters output
>> from this routine?
>>
>
> I thought (at the time) that it was sensible to add waters to a
> different chain ID to the protein atoms (I still do, in fact).   There
> are others (somewhat less involved in model-building I suspect) that
> think otherwise.
>
> Paul.
>
> ------------------------------
>
> Date:    Thu, 30 Jul 2009 20:56:14 +0200
> From:    Kay Diederichs <[log in to unmask]>
> Subject: Re: question of extra high B factor
>
> This is a cryptographically signed message in MIME format.
>
> --------------ms000208020902090005010407
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
> Content-Transfer-Encoding: 7bit
>
> Jiamu Du schrieb:
>> Dear All,
>> I am refining a structure of a complex between of 50kD protein and a
>> 20kD glycosylated protien. The data is of 2.9 A resolution. The  
>> wilson B
>> factor is as high as 86.3 A^2.
>> The refinement seems well with R/Rf of 0.21/0.25. But the B-factor is
>> extra high. For the 50kD part, the average B factor is 76.5 A^2.  
>> But the
>> B factor of the 20 kD glycosylated protein is as high as 133.3 A^2.
>> Although the electron density looks fine, even the sugar chain is  
>> seen
>> clearly.
>> My question is:
>> 1. How to reduce the B factor to a reasonable level?
>
> Please check out
> <http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Refinement#help.2C_my_protein_has_high_B-factors.21 
> >
>
>> 2. If it can not be redueced, when I published it, is this value  
>> acceptable?
>
> As there's nothing wrong with it, it can be published.
>
>> 3. In the same of similar resolutionIs, is there some other  
>> structures
>> like this situation?  A component or a subunit of the protein has a
>> extra high B factor as high as 130.
>
> Just look at the B-factors of new PDB entries which have a a  
> resolution
> worse than 3 A (e.g., membrane proteins) - there are quite a few of  
> those.
>
> HTH,
>
> Kay
> -- 
> Kay Diederichs                 http://strucbio.biologie.uni- 
> konstanz.de
> email: [log in to unmask]     Tel +49 7531 88 4049 Fax  
> 3183
> Fachbereich Biologie, Universitaet Konstanz, Box M647, D-78457  
> Konstanz
>
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> --------------ms000208020902090005010407--
>
> ------------------------------
>
> Date:    Thu, 30 Jul 2009 21:13:45 +0200
> From:    Luca Jovine <[log in to unmask]>
> Subject: Postdoctoral position in protein crystallography at  
> Karolinska Institutet
>
> POSTDOCTORAL FELLOWSHIP IN PROTEIN CRYSTALLOGRAPHY AT KAROLINSKA  
> INSTITUT=
> ET
>
> As part of a collaboration between the laboratories of Rune  
> Toftg=E5rd an=
> d Luca Jovine at the =
>
> KI Department of Biosciences and Nutrition and the Center for  
> Biosciences=
> =2C a postdoctoral =
>
> position is immediately available on structural studies of key  
> players in=
> the Hedgehog =
>
> signaling pathway=2E
>
> The Department of Biosciences and Nutrition has state-of-the-art  
> equipmen=
> t for protein =
>
> expression=2C purification and in house X-ray data collection=3B  
> frequent=
> access to synchrotron =
>
> sites and excellent computational facilities are also available=2E  
> With t=
> heir close integration of =
>
> academic=2C clinical and industrial research=2C KI and the Center  
> for Bio=
> sciences offer a highly =
>
> international and collaborative environment for biomedical studies=2E
>
> Highly motivated candidates with experience in protein expression  
> (partic=
> ularly in insect =
>
> and/or mammalian cells)=2C purification=2C crystallization and  
> structure =
> determination=2C are =
>
> encouraged to apply by e-mailing a curriculum vitae=2C summary of  
> researc=
> h experience and =
>
> interests=2C and names and contact information for two references to  
> Luca=
> Jovine =
>
> (luca=2Ejovine=40ki=2Ese)=2E Please note that=2C in order to be  
> eligible =
> for this scholarship=2C candidates =
>
> should be non-Swedish citizens who have obtained a doctorate or the  
> equiv=
> alent outside =
>
> Sweden=2E Deadline for application is 1 October 2009=2E
>
> ------------------------------------------------
> Luca Jovine=2C Ph=2ED=2E
> Group Leader=2C Protein Crystallography Unit
> Karolinska Institutet
> Department of Biosciences and Nutrition
> H=E4lsov=E4gen 7=2C SE-141 57 Huddinge=2C Sweden
> Voice=3A +46=2E(0)8=2E6083-301  FAX=3A +46=2E(0)8=2E6089-290
> E-mail=3A luca=2Ejovine=40ki=2Ese
> W3=3A http=3A//jovinelab=2Eorg
> ------------------------------------------------
>
> ------------------------------
>
> Date:    Thu, 30 Jul 2009 12:29:09 -0700
> From:    Donnie Berkholz <[log in to unmask]>
> Subject: Re: question of extra high B factor
>
> --/9DWx/yDrRhgMJTb
> Content-Type: text/plain; charset=us-ascii
> Content-Disposition: inline
> Content-Transfer-Encoding: quoted-printable
>
> On 00:00 Fri 31 Jul     , Jiamu Du wrote:
>> Dear All,
>> I am refining a structure of a complex between of 50kD protein and  
>> a 20kD
>> glycosylated protien. The data is of 2.9 A resolution. The wilson B  
>> factor
>> is as high as 86.3 A^2.
>> The refinement seems well with R/Rf of 0.21/0.25. But the B-factor  
>> is ext=
> ra
>> high. For the 50kD part, the average B factor is 76.5 A^2. But the  
>> B fact=
> or
>> of the 20 kD glycosylated protein is as high as 133.3 A^2. Although  
>> the
>> electron density looks fine, even the sugar chain is seen clearly.
>> My question is:
>> 1. How to reduce the B factor to a reasonable level?
>> 2. If it can not be redueced, when I published it, is this value  
>> acceptab=
> le?
>> 3. In the same of similar resolutionIs, is there some other  
>> structures li=
> ke
>> this situation?  A component or a subunit of the protein has a  
>> extra high=
> B
>> factor as high as 130.
>
> Our group published a paper a few years ago with an average B of 80  
> A^2=20
> (JMB 328:893 (2003)). One referee had some objections to this, so  
> we=20
> performed analyses of the whole Protein Data Bank for proteins at=20
> 2.5-3.0 A (see Fig. 2D in the paper). You may find this figure  
> useful if=20
> you need to convince anyone.
>
> --=20
> Thanks,
> Donnie
>
> Donnie Berkholz
> P. Andrew Karplus lab
> Oregon State University
>
> --/9DWx/yDrRhgMJTb
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>
> --/9DWx/yDrRhgMJTb--
>
> ------------------------------
>
> Date:    Thu, 30 Jul 2009 17:53:15 -0400
> From:    Jim Fairman <[log in to unmask]>
> Subject: Re: OpenGL Stereo 3D on 120 Hz LCDs, at last!
>
> --0015174be078b3c278046ff354f1
> Content-Type: text/plain; charset=ISO-8859-1
> Content-Transfer-Encoding: 7bit
>
> Just got this working on a machine with a Quadro FX 3800.  Stereo  
> looks
> great on both Pymol (v1.1) and the latest build of WinCoot (v0.6 build
> 2172).
>
> On Fri, Jul 17, 2009 at 1:20 AM, Warren DeLano <[log in to unmask]>  
> wrote:
>
>> FYI, for folks not subscribed to pymol-users:
>>
>>
>>
>> nVidia today released beta drivers which at last enable OpenGL- 
>> based stereo
>> 3D visualization on 120 Hz LCDs using Quadro graphics cards.  So  
>> long as you
>> are willing to put up with Windows, you can finally abandon those  
>> old CRTs
>> without spending a fortune and without sacrificing quality of the  
>> stereo 3D
>> effect.
>>
>>
>>
>> Details posted at http://www.pymol.org
>>
>>
>>
>> Cheers,
>>
>> Warren
>>
>>
>>
>
>
>
> -- 
> Jim Fairman
> Graduate Research Assistant
> Department of Biochemistry, Cellular, and Molecular Biology (BCMB)
> University of Tennessee -- Knoxville
> 216-368-3337 [log in to unmask] [log in to unmask]
>
> --0015174be078b3c278046ff354f1
> Content-Type: text/html; charset=ISO-8859-1
> Content-Transfer-Encoding: quoted-printable
>
> Just got this working on a machine with a Quadro FX 3800.=A0 Stereo  
> looks g=
> reat on both Pymol (v1.1) and the latest build of WinCoot (v0.6  
> build 2172)=
> .<br><br><div class=3D"gmail_quote">On Fri, Jul 17, 2009 at 1:20 AM,  
> Warren=
> DeLano <span dir=3D"ltr">&lt;<a  
> href=3D"mailto:[log in to unmask]">warren@d=
> elsci.com</a>&gt;</span> wrote:<br>
> <blockquote class=3D"gmail_quote" style=3D"border-left: 1px solid  
> rgb(204, =
> 204, 204); margin: 0pt 0pt 0pt 0.8ex; padding-left: 1ex;">
>
>
>
>
>
>
>
>
>
>
>
> <div link=3D"blue" vlink=3D"purple" lang=3D"EN-US">
>
> <div>
>
> <p><font size=3D"2" face=3D"Arial"><span style=3D"font-size: 10pt;  
> font-fam=
> ily: Arial;">FYI, for folks not subscribed to pymol-users:=A0 </ 
> span></font=
>> </p>
>
> <p><font size=3D"2" face=3D"Arial"><span style=3D"font-size: 10pt;  
> font-fam=
> ily: Arial;">=A0</span></font></p>
>
> <p><font size=3D"2" face=3D"Arial"><span style=3D"font-size: 10pt;  
> font-fam=
> ily: Arial;">nVidia today released beta drivers which at last enable  
> OpenGL=
> -based
> stereo 3D visualization on 120 Hz LCDs using Quadro graphics  
> cards.=A0 So l=
> ong
> as you are willing to put up with Windows, you can finally abandon  
> those ol=
> d CRTs
> without spending a fortune and without sacrificing quality of the  
> stereo 3D
> effect.</span></font></p>
>
> <p><font size=3D"2" face=3D"Arial"><span style=3D"font-size: 10pt;  
> font-fam=
> ily: Arial;">=A0</span></font></p>
>
> <p><font size=3D"2" face=3D"Arial"><span style=3D"font-size: 10pt;  
> font-fam=
> ily: Arial;">Details posted at <a href=3D"http://www.pymol.org/"  
> target=3D"=
> _blank">http://www.pymol.org</a></span></font></p>
>
> <p><font size=3D"2" face=3D"Arial"><span style=3D"font-size: 10pt;  
> font-fam=
> ily: Arial;">=A0</span></font></p>
>
> <p><font size=3D"2" face=3D"Arial"><span style=3D"font-size: 10pt;  
> font-fam=
> ily: Arial;">Cheers,</span></font></p>
>
> <p><font size=3D"2" face=3D"Arial"><span style=3D"font-size: 10pt;  
> font-fam=
> ily: Arial;">Warren</span></font><font size=3D"2"  
> face=3D"Arial"><span styl=
> e=3D"font-size: 10pt; font-family: Arial;"></span></font></p>
>
> <p><font size=3D"2" face=3D"Arial"><span style=3D"font-size: 10pt;  
> font-fam=
> ily: Arial;">=A0</span></font></p>
>
> </div>
>
> </div>
>
>
> </blockquote></div><br><br clear=3D"all"><br>-- <br>Jim  
> Fairman<br>Graduate=
> Research Assistant<br>Department of Biochemistry, Cellular, and  
> Molecular =
> Biology (BCMB)<br>University of Tennessee --  
> Knoxville<br>216-368-3337 <a h=
> ref=3D"mailto:[log in to unmask]">[log in to unmask]</a> <a href=3D"mailto:jame=
> [log in to unmask]">[log in to unmask]</a><br>
>
>
> --0015174be078b3c278046ff354f1--
>
> ------------------------------
>
> Date:    Thu, 30 Jul 2009 15:23:04 -0700
> From:    James Holton <[log in to unmask]>
> Subject: Re: Foils for energy calibration
>
> At ALS, we have a box of foils from EXAFS Materials that seems to  
> get=20
> passed around from beamline to beamline.  I ran absorption scans on  
> 17=20
> edges from the metals in the box one day, and found that there was=20
> considerable scatter in the expected vs observed edge positions:
> http://bl831.als.lbl.gov/~jamesh/pickup/mono_calib.png
> Here I have plotted the "correct" position of each edge as  
> determined by=20
> Bearden & Burr (1967), against the edge I determined using the  
> criterion=20
> recommended in the "Reference Spectra" document in the=20
> exafsmaterials.com website: the first inflection point in the  
> derivative=20
> spectrum.  I think a large amount of the scatter is because my mono=20
> (like many PX/MX beamlines) is Si(111) and not Si(220) like the one  
> used=20
> to determine the reference spectra.  It is not hard to imagine how=20
> blurring the spectrum with a wider energy spread of the incident  
> beam=20
> will shift the position of the "edge".  One could try to use the=20
> electron binding energy tabulated in the "little orange book":
> http://xdb.lbl.gov/Section1/Sec_1-1.html
> but these do not always take into account the "near edge" features  
> (like=20
> the white line from SeMet) which change depending on the chemical=20
> environment around the metal, radiation damage, etc.  It would be  
> nice=20
> if someone could calibrate some standard reference materials using  
> a=20
> Si(111) monochromator, but I don't know of anyone who has done this.
>
>
> However, another way to get your x-ray wavelength is using Bragg's  
> law:
> lambda =3D 2*d*sin(theta)
> and the d-spacing of silicon is known to be 5.43159 =B1 0.00020 A,  
> and=20
> NIST will sell you certified Si powder:
> https://www-s.nist.gov/srmors/view_detail.cfm?srm=3D640d
>
> The problem here is that although you know d very accurately, the  
> error=20
> in lambda is dominated by sin(theta), or rather the uncertainty in  
> your=20
> detector distance.  The pixel field on most detectors is actually  
> quite=20
> accurate, as a NIST-traceable calibration is used to make the  
> pinhole=20
> calibration mask, and the encoder on most detector distance stages  
> is=20
> very accurate for relative moves (counting ticks on the encoder).   
> But=20
> there is always an offset from the "zero" position predicted by the=20
> encoder to the true center of rotation that is hard to know.=20
>
> Nevertheless, all you really want is for the d-spacing of silicon  
> powder=20
> rings to be right at all detector distances. You can use the  
> program=20
> FIT2D to refine the wavelength, distance, detector tilt, etc. or  
> any=20
> combination thereof for a given image, but you will find that the=20
> repeatability of such a fit (using different starting parameters) is  
> not=20
> great because the distance and wavelength are highly correlated. =20
> However, there is a way around this:
>
> Since we know that a relative move of the distance will be  
> accurate,=20
> there should be one and only one offset that you can add to the  
> recorded=20
> value of distance of each image to make it the "right" distance.   
> You=20
> can define the "right" offset as the one where FIXing the resulting=20
> "right" distance in FIT2D and refining everything else gives you  
> the=20
> same refined value for the wavelength from every image.  You need  
> to=20
> manually "refine" this offset for a few rounds.  What you will  
> generally=20
> see is that the graph of fitted wavelength vs the distance is a  
> straight=20
> line, and you want to make the slope of this line to be zero. =20
> Eventually, you will arrive at some offset that gives you the  
> smallest=20
> spread in refined wavelength values.  The average refined wavelength  
> is=20
> then the "true" wavelength.  Should be able to get it within one or  
> two=20
> eV.  Perhaps more if you take a lot of silicon powder images.
>
> At ALS beamlines 8.3.1 and 12.3.1 I have done both kinds of  
> calibration,=20
> and I am fairly certain I get the wavelength accurate to within 1 eV  
> by=20
> calibrating the half-way-up point of an absorption scan of a ~122  
> micron=20
> thick copper metal foil to 8979.0 eV.
>
> -James Holton
> MAD Scientist
>
>
> Richard Gillilan wrote:
>> In the past we've used elemental foils from exafsmaterials.com for=20
>> energy calibration of our MAD beamline. These standards are for  
>> EXAFS=20
>> and XANES. Most are thin (5 micron) metal foils.
>>
>> Has anyone had experience with other sources of standards or other=20
>> forms (such as compounds rather than pure elements)?
>>
>> I notice that a number of companies offer XRF standard kits.
>>
>> Richard Gillilan
>> MacCHESS
>
> ------------------------------
>
> End of CCP4BB Digest - 29 Jul 2009 to 30 Jul 2009 (#2009-207)
> *************************************************************