Re: [ccp4bb] No improvement in R-factor after Refmac.

[CRAIG A BINGMAN]

You really need to approach such situations with caution.  Examination of the 
relatively small number of axial reflections probably show that there might be 
twofold screw axes in all three directions.  But a non-crystallographic 
microscopic translation of nearly 0.5 in the direction of a crystallographic 
axis will give the same pattern of strong and weak reflections as a 
crystallographic twofold screw axis.  If I were you, I would be very sure to 
try molecular replacement in all possible orthorhombic space groups.  Several 
programs, including Phaser, will organize that exhaustive search across all 
eight possibilities for you.

On Mar 17, 2017, at 11:56 PM, Polisetty Satya Dev 
mailto:pvss...@gmail.com>> wrote:

Hi,

We checked all possible space groups of orthorhombic crystal system using Scala 
and Pointless but the statistics show that P212121 is the possible space group.

Thank You,
Satya Dev

On Fri, Mar 17, 2017 at 8:03 PM, Teplyakov, Alexey [JRDUS] 
mailto:atepl...@its.jnj.com>> wrote:
Check the space group. It may be orthorhombic with a pure rotational axis (e.g. 
P21212) or even monoclinic.

From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Polisetty Satya Dev
Sent: Friday, March 17, 2017 9:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] [ccp4bb] No improvement in R-factor after Refmac.

Dear all,
I solved a structure at 2.0 A resolution with R-work and R-free values 0.25 and 
0.32 respectively and I am stuck at Refmac step where there is no further 
reduction in R-factor.

The above stated values were obtained after several rounds of manual refinement 
followed by refmac. There are also areas where electron density is missing 
around peptide backbone in one of the monomer in ASU.

Can anyone please tell me how can I improve the electron density and R-factor.

The structure solution was obtained using Phaser MR and here are the data 
statistics:

Average unit cell: 81.95, 100.40, 156.96,   90.00, 90.00, 90.00,
Space group: P212121,
Completeness 99.5,
Multiplicity 6.4,
Four monomers per ASU.
Solvent content: 47%.

Thank you everyone,
Satya Dev,
JNCASR.

Re: [ccp4bb] No improvement in R-factor after Refmac.

[Polisetty Satya Dev]

Hi,

We ran Scala program with 2 A resolution cut-off and it gave mean I / sig I
value as follows

Overall: 7.3   OuterShell: 2.0 InnerShell: 16.5

CC(1/2) : Overall: 0.994   OuterShell: 0.665 InnerShell: 0.997

We also tried Scala with 2.33 A resolution cut-off and the statistics are
as follows

Mean I / sig I : Overall: 11.3   OuterShell: 5.0 InnerShell: 20.0

CC(1/2) : Overall: 0.995   OuterShell: 0.942 InnerShell: 0.997

Thank You,
Satya Dev.

On Fri, Mar 17, 2017 at 7:59 PM, Mohamed Noor  wrote:

> How do you know it's really 2 A resolution? How is your CC(1/2)?
>
>
> On 17/03/2017 13:51, Polisetty Satya Dev wrote:
>
> Dear all,
>
> I solved a structure at 2.0 A resolution with R-work and R-free values
> 0.25 and 0.32 respectively and I am stuck at Refmac step where there is no
> further reduction in R-factor.
>
> The above stated values were obtained after several rounds of manual
> refinement followed by refmac. There are also areas where electron density
> is missing around peptide backbone in one of the monomer in ASU.
>
> Can anyone please tell me how can I improve the electron density and
> R-factor.
>
> The structure solution was obtained using Phaser MR and here are the data
> statistics:
>
> Average unit cell: 81.95, 100.40, 156.96,   90.00, 90.00, 90.00,
>
> Space group: P212121,
>
> Completeness 99.5,
>
> Multiplicity 6.4,
>
> Four monomers per ASU.
>
> Solvent content: 47%.
>
>
> Thank you everyone,
>
> Satya Dev,
>
> JNCASR.
>
>
>

Re: [ccp4bb] No improvement in R-factor after Refmac.

[Polisetty Satya Dev]

Hi,

We checked all possible space groups of orthorhombic crystal system using
Scala and Pointless but the statistics show that P212121 is the possible
space group.

Thank You,
Satya Dev

On Fri, Mar 17, 2017 at 8:03 PM, Teplyakov, Alexey [JRDUS] <
atepl...@its.jnj.com> wrote:

> Check the space group. It may be orthorhombic with a pure rotational axis
> (e.g. P21212) or even monoclinic.
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Polisetty
> Satya Dev
> *Sent:* Friday, March 17, 2017 9:51 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [EXTERNAL] [ccp4bb] No improvement in R-factor after Refmac.
>
>
>
> Dear all,
>
> I solved a structure at 2.0 A resolution with R-work and R-free values
> 0.25 and 0.32 respectively and I am stuck at Refmac step where there is no
> further reduction in R-factor.
>
> The above stated values were obtained after several rounds of manual
> refinement followed by refmac. There are also areas where electron density
> is missing around peptide backbone in one of the monomer in ASU.
>
> Can anyone please tell me how can I improve the electron density and
> R-factor.
>
>
> The structure solution was obtained using Phaser MR and here are the data
> statistics:
>
>
>
> Average unit cell: 81.95, 100.40, 156.96,   90.00, 90.00, 90.00,
>
> Space group: P212121,
>
> Completeness 99.5,
>
> Multiplicity 6.4,
>
> Four monomers per ASU.
>
> Solvent content: 47%.
>
>
>
> Thank you everyone,
>
> Satya Dev,
>
> JNCASR.
>

Re: [ccp4bb] [EXTERNAL] [ccp4bb] Pymol alignment command grammar help

[Yong Wang]

The “name” means atom names, e.g. n for nitrogen (backbone), yes, you would use 
“name” (or n. in short) in your command.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Alex Lee
Sent: Friday, March 17, 2017 2:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] [ccp4bb] Pymol alignment command grammar help

Dear CCP4BB members,

I tried to use pymol command to align two proteins, I read the pymol wiki and I 
could not understand the command grammars (I am not computer major and not 
familiar with machine language).

For example pymol wiki says as below:

Furthermore, you may wish to restrict the alignment to just the backbone atoms, 
so you can say:

align structure2 and resi 1-100 and name n+ca+c+o, structure1 and resi 300-400 
and name n+ca+c+o

or in short form:
align structure2 & i. 1-100 & n. n+ca+c+o, structure1 & i. 300-400 & n. n+ca+c+o


My questions are: 1. what does "name" mean in the command, should I just type 
"name" as is in the pymol command?

2. If I want to align two conserved helix of a protein dimer (each helix is on 
one monomer) to its single point mutant (mutation is not in the conserved helix 
region), as the helix is on different protein chains, could I write one command 
as: align structure 2 and chain X and resi xxx and chain Y and resi xxx and 
name ca, structure 1 and chain X and resi xxx and chain Y and resi xxx and name 
ca?



Thanks for any input on this.

[ccp4bb] Pymol alignment command grammar help

[Alex Lee]

Dear CCP4BB members,

I tried to use pymol command to align two proteins, I read the pymol wiki
and I could not understand the command grammars (I am not computer major
and not familiar with machine language).

For example pymol wiki says as below:

Furthermore, you may wish to restrict the alignment to just the backbone
atoms, so you can say:

align structure2 and resi 1-100 and name n+ca+c+o, structure1 and resi
300-400 and name n+ca+c+o

or in short form:

align structure2 & i. 1-100 & n. n+ca+c+o, structure1 & i. 300-400 & n.
n+ca+c+o


My questions are: 1. what does "name" mean in the command, should I just
type "name" as is in the pymol command?

2. If I want to align two conserved helix of a protein dimer (each helix is
on one monomer) to its single point mutant (mutation is not in the
conserved helix region), as the helix is on different protein chains, could
I write one command as: align structure 2 and chain X and resi xxx and
chain Y and resi xxx and name ca, structure 1 and chain X and resi xxx and
chain Y and resi xxx and name ca?



Thanks for any input on this.

Re: [ccp4bb] ITC question

[Scott Horowitz]

I recall a case of this a few years ago where it had to do with the
relative concentration of the protein to the buffer/salt molecules (can't
remember which anymore), which ended up being important in the binding that
was observed. So it was entirely a concentration effect that caused the
difference, where using the higher concentrations for ITC caused an
increase in the observed Kd.

Scott

On Fri, Mar 17, 2017 at 1:47 PM, DUMAS Philippe (VIE) <
p.du...@ibmc-cnrs.unistra.fr> wrote:

>
> Le Vendredi 17 Mars 2017 16:07 CET, Nicholas Larsen  H3BIOMEDICINE.COM> a écrit:
>
> Do you mean that the affinity from ITC is 100-fold weaker ? Which would
> mean that the Kd values from ITC are 100-fold larger (in the range 0.1-1
> µM) ?
> If this is the case, it makes me think to a problem that we have observed
> by comparing Kd values obtained by ITC and by Mass Spec. The Kd values by
> mass spec were very well determined and smaller (higher affinity) than
> those obtained by ITC after a classical processing. It turns out that
> processing the ITC data with two competing modes of binding revealed the
> correct higher affinity binding mode observed by Mass Spec (80 %) mixed
> with the lower-affinity binding mode (20 %). The a priori very strange
> thing, which we finally explained, is that the initial processing of the
> ITC data with only one binding mode had mixed wrongly the Kd for the
> low-affinity binding mode, but the DeltaH for the  high-affinity binding
> mode.
>
> I suggest that you have a look to our paper: Wolff et al., J Am Soc Mass
> Spectrom. 2017; 28(2): 347–357 (Open access).
> I hope it will help.
> Philippe Dumas
>
> > Dear colleagues,
> > We have a target where people have measured Kd's for ligands using
> > radioligand binding assays.  Several publications report Kd's of single
> > digit nanomolar and we are able to reproduce that data using this assay
> > format.  When we try to do the same measurement using ITC, we generate
> > beautiful data, but the Kd's from ITC are at least 100-fold weaker.  Does
> > anyone have a suggestion how to reconcile this huge difference?
> >
> > SPR studies show the ligands have a very long residence time, so one
> thing
> > I wondered is if ITC can underestimate a Kd if the off-rate is on the
> order
> > of minutes-hours.  Is this a reasonable explanation?
> >
> > Please, any other ideas are welcome.
> > Best,
> > Nick
> >
> > --
> > [This e-mail message may contain privileged, confidential and/or
> > proprietary information of H3 Biomedicine. If you believe that it has
> been
> > sent to you in error, please contact the sender immediately and delete
> the
> > message including any attachments, without copying, using, or
> distributing
> > any of the information contained therein. This e-mail message should not
> be
> > interpreted to include a digital or electronic signature that can be used
> > to authenticate an agreement, contract or other legal document, nor to
> > reflect an intention to be bound to any legally-binding agreement or
> > contract.]
>
>
>
>
>


-- 
Scott Horowitz, Ph.D.
Postdoctoral Fellow

University of Michigan
Department of Molecular, Cellular, and Developmental Biology
Bardwell lab
830 N. University Ave, Room 4007
Ann Arbor, MI 48109
phone: 734-647-6683
fax: 734-615-4226

Re: [ccp4bb] ITC question

[VIE]

Le Vendredi 17 Mars 2017 16:07 CET, Nicholas Larsen 
 a écrit:

Do you mean that the affinity from ITC is 100-fold weaker ? Which would mean 
that the Kd values from ITC are 100-fold larger (in the range 0.1-1 µM) ?
If this is the case, it makes me think to a problem that we have observed by 
comparing Kd values obtained by ITC and by Mass Spec. The Kd values by mass 
spec were very well determined and smaller (higher affinity) than those 
obtained by ITC after a classical processing. It turns out that processing the 
ITC data with two competing modes of binding revealed the correct higher 
affinity binding mode observed by Mass Spec (80 %) mixed with the 
lower-affinity binding mode (20 %). The a priori very strange thing, which we 
finally explained, is that the initial processing of the ITC data with only one 
binding mode had mixed wrongly the Kd for the low-affinity binding mode, but 
the DeltaH for the  high-affinity binding mode.

I suggest that you have a look to our paper: Wolff et al., J Am Soc Mass 
Spectrom. 2017; 28(2): 347–357 (Open access).
I hope it will help.
Philippe Dumas

> Dear colleagues,
> We have a target where people have measured Kd's for ligands using
> radioligand binding assays.  Several publications report Kd's of single
> digit nanomolar and we are able to reproduce that data using this assay
> format.  When we try to do the same measurement using ITC, we generate
> beautiful data, but the Kd's from ITC are at least 100-fold weaker.  Does
> anyone have a suggestion how to reconcile this huge difference?
>
> SPR studies show the ligands have a very long residence time, so one thing
> I wondered is if ITC can underestimate a Kd if the off-rate is on the order
> of minutes-hours.  Is this a reasonable explanation?
>
> Please, any other ideas are welcome.
> Best,
> Nick
>
> --
> [This e-mail message may contain privileged, confidential and/or
> proprietary information of H3 Biomedicine. If you believe that it has been
> sent to you in error, please contact the sender immediately and delete the
> message including any attachments, without copying, using, or distributing
> any of the information contained therein. This e-mail message should not be
> interpreted to include a digital or electronic signature that can be used
> to authenticate an agreement, contract or other legal document, nor to
> reflect an intention to be bound to any legally-binding agreement or 
> contract.]

[ccp4bb] postdoctoral positions at Cold Spring Harbor Laboratory

[Joshua-Tor, Leemor]

Applications are invited for postdoctoral positions in my lab at Cold Spring 
Harbor Laboratory to work on the structure and mechanism of gene silencing 
pathways or DNA replication.
Please see our web page for more details 
(http://joshua-torlab.labsites.cshl.edu/).

Positions are available for highly motivated, outstanding applicants with a 
recent PhD and experience in electron microscopy, protein crystallography, or a 
related field, or applicants with experience in biochemistry and molecular 
biology with a keen interest in structural biology.

We are about to take delivery of a Titan Krios cryo electron microscope. 
Combined with the nearby NSLS-II coming on line, these are exciting times with 
exciting capabilities.

Cold Spring Harbor Laboratory (CSHL) provides a unique, stimulating and very 
collaborative environment for scientific interactions. Located on the North 
Shore of Long Island, CSHL is in a beautiful setting and is a wonderful place 
to expand one’s knowledge of biology in many different areas, particularly via 
CSHL’s internationally acclaimed Meetings and Conferences, which our postdocs 
can freely attend. For details about CSHL please see our web site at: 
http://www.cshl.edu/

Please send CV, list of publications, a summary of research experience and 
interests, and contact information of three references.

--
Leemor Joshua-Tor, Ph. D.
HHMI Investigator
Professor, Watson School of Biological Sciences
Cold Spring Harbor Laboratory
1 Bungtown Road
Cold Spring Harbor, NY 11724
p: (516) 367-8821
f: (516) 367-8873
e: lee...@cshl.edu
w: 
http://joshua-torlab.labsites.cshl.edu

Re: [ccp4bb] Positive densities map on selective residues on Coot or Pymol

[Xiao Lei]

Dear All,

Thanks for the information.
I tried the way suggested by pymol wiki, but pymol fail to display the map.
This is what I did: Run Fft to generate simple mapin ccp4i, input mtz map
from refmac, set F1=FWT and PHIC=PHWT, Sigma=SigF, Weight=FOM.  Add the
file extension .map.ccp4 to the generated output map. After open by pymol,
only a unit cell sign is shown (I attach here), no map is displayed.. I use
Pymol 1.8.X in Win7.

Any further input is appreciated.



.




On Fri, Mar 17, 2017 at 7:41 AM, Xiao Lei  wrote:

> Dear All,
>
> In Coot, I could adjust the densities of map (2Fo-Fc in my case) radius,
> but I'd like to show the density on selective residues, not on the
> unselected part of structure, is there a way to do it? I am using WinCoot
> 0.81.
>
> In addition, could Pymol do it?
>
> Thanks ahead!
>

[ccp4bb] ITC question

[Nicholas Larsen]

Dear colleagues,
We have a target where people have measured Kd's for ligands using
radioligand binding assays.  Several publications report Kd's of single
digit nanomolar and we are able to reproduce that data using this assay
format.  When we try to do the same measurement using ITC, we generate
beautiful data, but the Kd's from ITC are at least 100-fold weaker.  Does
anyone have a suggestion how to reconcile this huge difference?

SPR studies show the ligands have a very long residence time, so one thing
I wondered is if ITC can underestimate a Kd if the off-rate is on the order
of minutes-hours.  Is this a reasonable explanation?

Please, any other ideas are welcome.
Best,
Nick

-- 
[This e-mail message may contain privileged, confidential and/or 
proprietary information of H3 Biomedicine. If you believe that it has been 
sent to you in error, please contact the sender immediately and delete the 
message including any attachments, without copying, using, or distributing 
any of the information contained therein. This e-mail message should not be 
interpreted to include a digital or electronic signature that can be used 
to authenticate an agreement, contract or other legal document, nor to 
reflect an intention to be bound to any legally-binding agreement or 
contract.]

[ccp4bb] Positive densities map on selective residues on Coot or Pymol

[Xiao Lei]

Dear All,

In Coot, I could adjust the densities of map (2Fo-Fc in my case) radius,
but I'd like to show the density on selective residues, not on the
unselected part of structure, is there a way to do it? I am using WinCoot
0.81.

In addition, could Pymol do it?

Thanks ahead!

Re: [ccp4bb] No improvement in R-factor after Refmac.

[Teplyakov, Alexey [JRDUS]]

Check the space group. It may be orthorhombic with a pure rotational axis (e.g. 
P21212) or even monoclinic.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Polisetty 
Satya Dev
Sent: Friday, March 17, 2017 9:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] [ccp4bb] No improvement in R-factor after Refmac.

Dear all,
I solved a structure at 2.0 A resolution with R-work and R-free values 0.25 and 
0.32 respectively and I am stuck at Refmac step where there is no further 
reduction in R-factor.

The above stated values were obtained after several rounds of manual refinement 
followed by refmac. There are also areas where electron density is missing 
around peptide backbone in one of the monomer in ASU.

Can anyone please tell me how can I improve the electron density and R-factor.

The structure solution was obtained using Phaser MR and here are the data 
statistics:


Average unit cell: 81.95, 100.40, 156.96,   90.00, 90.00, 90.00,

Space group: P212121,

Completeness 99.5,

Multiplicity 6.4,

Four monomers per ASU.

Solvent content: 47%.



Thank you everyone,

Satya Dev,

JNCASR.

Re: [ccp4bb] No improvement in R-factor after Refmac.

[Masooma Rasheed]

One way is to refine the best chain out of four, first and then generate a
seperate pdb for that chain only and use molecular replacement to find the
rest of the three chains. It does help with improving map to certain
extent. Also delete the missing residues one by one and then run refmac to
see if get any green density back for them to rebuild them in the right
conformation..

On Fri, Mar 17, 2017 at 1:51 PM, Polisetty Satya Dev 
wrote:

> Dear all,
>
> I solved a structure at 2.0 A resolution with R-work and R-free values
> 0.25 and 0.32 respectively and I am stuck at Refmac step where there is no
> further reduction in R-factor.
>
> The above stated values were obtained after several rounds of manual
> refinement followed by refmac. There are also areas where electron density
> is missing around peptide backbone in one of the monomer in ASU.
>
> Can anyone please tell me how can I improve the electron density and
> R-factor.
>
> The structure solution was obtained using Phaser MR and here are the data
> statistics:
>
> Average unit cell: 81.95, 100.40, 156.96,   90.00, 90.00, 90.00,
>
> Space group: P212121,
>
> Completeness 99.5,
>
> Multiplicity 6.4,
>
> Four monomers per ASU.
>
> Solvent content: 47%.
>
>
> Thank you everyone,
>
> Satya Dev,
>
> JNCASR.
>
>


-- 
Dr. Masooma Rasheed
Division of Molecular Biosciences
Biochemistry Building Level 5
Imperial College London
South Kensington
London SW7 2AZ
UK

[ccp4bb] No improvement in R-factor after Refmac.

[Polisetty Satya Dev]

Dear all,

I solved a structure at 2.0 A resolution with R-work and R-free values 0.25
and 0.32 respectively and I am stuck at Refmac step where there is no
further reduction in R-factor.

The above stated values were obtained after several rounds of manual
refinement followed by refmac. There are also areas where electron density
is missing around peptide backbone in one of the monomer in ASU.

Can anyone please tell me how can I improve the electron density and
R-factor.

The structure solution was obtained using Phaser MR and here are the data
statistics:

Average unit cell: 81.95, 100.40, 156.96,   90.00, 90.00, 90.00,

Space group: P212121,

Completeness 99.5,

Multiplicity 6.4,

Four monomers per ASU.

Solvent content: 47%.


Thank you everyone,

Satya Dev,

JNCASR.

[ccp4bb] Research assistant postion Heidelberg

[Balendu Avvaru]

A research assistant position is available immediately at the BioMedX 
Innovation Center in Heidelberg, Germany. The ideal candidate ought to have a 
background in protein biochemistry or microbiology, although other biological 
fields are not deal breakers. Local candidates will be given preference. Please 
send applications to avv...@bio.mx  More information on the position is 
available at http://apply.bio.mx/job/2017-J08

Balendu Avvaru
Group Leader
BioMedX Innovation Center
Heidelberg, Germany