[ccp4bb] Postdoctoral researcher position at The Walter & Eliza Hall Institute

[Richard Birkinshaw]

Posted on behalf of Alan F. Cowman:

We are hiring a protein chemist/structural biologist. Please see below or click 
on link below for other details.

https://www.wehi.edu.au/research-officer-cowman-laboratory


About the position

The postdoctoral scientist who fills this position will be an expert in protein 
science and structural biology to work as part of a team within the Cowman 
laboratory to express proteins in a functional form. The position requires 
experience in protein expression, purification and characterization and a 
strong background in structural biology. The appointee will collaborate within 
a team environment on a project funded by the Wellcome Trust and in 
collaboration with Merck USA that is aimed towards the development of new 
antimalarial drugs.
Key Responsibilities
Technical Service – Protein Science
· Design and assemble DNA constructs for protein expression in 
bacteria, yeast, insect and mammalian systems.
· Express and purify proteins in bacteria, yeast, insect and mammalian 
cells.
· Characterize recombinant proteins (MS, ITC, SPR, CD, enzyme kinetics 
etc.)
· Crystallize proteins for structural studies by X-ray diffraction.
General
· Accurate and timely completion of tasks to enable the project to meet 
deadlines
· Source and read the literature relevant to the project
· Composition of figures and methods data for publications.
· Carefully maintain laboratory records with a particular awareness of 
IP issues
· Assist in maintaining the laboratory in a clean, well-stocked, safe 
and orderly state.


Selection Criteria
Personal qualities

· Strong work ethic, capacity to work independently or as part of a 
team, ability to troubleshoot experimental problems, willingness to aid 
students, good record-keeping with meticulous attention to detail.

Knowledge and skills

· PhD in protein science (biochemistry, structural biology, synthetic 
biology etc.)

· Experience in protein expression (bacterial and eukaryotic systems), 
purification (IMAC, SEC, IEX, HIC), and characterization (MS, ITC, SPR, CD, 
enzyme kinetics etc).

· Experience in determining protein structures by X-ray crystallography.

· Sound communication skills (both oral and written English)

Terms of appointment

The position is available for a period of 2 years in the first instance. Up to 
17% superannuation and very attractive salary packaging options are available.

A position description is 
available.

General enquiries can be directed to Professor. Alan 
Cowman.



How to apply
Please email your application including cover letter, CV and a letter 
addressing the key selection criteria in pdf format to 
jobapplicati...@wehi.edu.au
 quoting reference WEHI/CAAC in the subject line.

At the Walter and Eliza Hall Institute we strive to ensure our staff and 
students enjoy a great working environment.  We value diversity and gender 
equity in our work 
force and promote flexible working arrangements for staff to balance working 
requirements and personal needs. We have implemented a number of gender equity 
initiatives to support female laboratory heads.





Alan F. Cowman  FAA FRS
Deputy Director – Science Strategy
Head, Division of Infection and Immunity
The Walter and Eliza Hall institute of Medical Research

Professor, Department of Medical Biology
Faculty of Medicine, Dentistry and Health Sciences
The University of Melbourne

Postal Address:
1G Royal Parade
Parkville, VIC 3052
Australia

Contact:
cow...@wehi.edu.au
Telephone: 61-3-93452446
Mobile: 61-430141209

---I'm an Athena SWAN Advocate. I call out sexism and promote equality.---

Re: [ccp4bb] (arcane) How to generate complete set of indices at low res

[James Holton]

I say "putative" because I don't know what your space group is.

In P212121 the reflection h,k,l = 0,0,1 is absent, but in P222 it is not 
absent.  So, if your unit cell is a=30 b=40 c=60 the lowest-angle hkl 
you will get is at 60 A resolution (0,0,1) in P222, but the lowest-angle 
reflection you will get out of P212121 will be (0,1,1), at 33.3 A 
resolution.  This is because 0,1,0 is also absent.  So, if you ever 
specify P212121 in your pipeline the 0,0,1 reflection will be lost.  
Same thing happens with most any screw-vs-rotation axis assignment.


You loose other reflections to absences too, of course, but the 
lowest-order ones have an annoying habit of defining the "resolution 
range", and this can sometimes get set at one point in the pipeline and 
applied to subsequent operations, even if you change the space group 
back.  This could also be happening to you?


It is also possible to a subtle change in unit cell can move your 
lowest-order (and also the highest order) reflections across the defined 
"resolution range" boundaries.  Sometimes even round-off error can be 
enough.


So, if low-resolution is important it is always a good idea to replace 
the low-angle resolution limit with  A.  Just be sure your beamstop 
was properly masked off.


-James Holton
MAD Scientist


On 4/5/2018 10:55 AM, Pearce, N.M. (Nick) wrote:
Could you expand a bit on what you mean by a “putative” systematic 
absence? (e.g. why only the lowest order hkl?)




On 5 Apr 2018, at 19:39, James Holton > wrote:


You need to be careful with the exact space group at the particular 
stage in your pipeline here. Often the lowest-order hkl is a putative 
systematic absence, so if you uniqueify in P222 you will get it, but 
if you uniqueify in P212121, then you won't.  That sort of thing.  
Note that it doesn't matter what the "true" space group is, it only 
matters what is in the mtz header when you run uniqueify.


Could that be what is going on?

-James Holton

MAD Scisntist


On 4/5/2018 3:52 AM, Frank von Delft wrote:


Hello - can anybody shed light on this mystery:

We need (for PanDDA analysis) a lot of datasets each to have the 
complete set of low resolution indices, whether measured or not. 
(Refmac adds the estimates as DFc, which is crucial when comparing 
maps.)


In ccp4, there are two obvious ways to get these indices complete:

  * uniqueify
  * CAD using the keyword "RESOLUTION FILE 1 999 "  (999 is
the low resolution limit).

Mystifyingly, in ~1% of datasets, one or the other route misses one 
or two indices.  Our work-around is to go belt-and-braces and run 
both for each dataset.



It does however remain a bug.  Does anybody have any idea what's 
happening?  We can send example datasets to any volunteers who want 
to fiddle with it.


phx

Re: [ccp4bb] B-factor standardization

[Pavel Afonine]

> If I am not wrong, I remember that someone proposed to standardize
> B-factors of protein atoms as “BS = B - Bave”, where Bave is the average
> B-factor of the protein.


This will make some of BS negative (if B

Re: [ccp4bb] (arcane) How to generate complete set of indices at low res

[Pavel Afonine]

Just in case you find it helpful, you can get 100% complete set of
reflections (Fcalc) in specified resolution range using

phenix.fmodel model.pdb high_res=2.5 low_res=15

or if you leave out low_res it will go all the way up to theoretical limit
of low resolution.

If you have/use cctbx then I can show to do this at lower (Python) level.

Pavel

On Thu, Apr 5, 2018 at 3:52 AM, Frank von Delft  wrote:

> Hello - can anybody shed light on this mystery:
>
> We need (for PanDDA analysis) a lot of datasets each to have the complete
> set of low resolution indices, whether measured or not.  (Refmac adds the
> estimates as DFc, which is crucial when comparing maps.)
>
> In ccp4, there are two obvious ways to get these indices complete:
>
>- uniqueify
>- CAD using the keyword "RESOLUTION FILE 1 999 "  (999 is the
>low resolution limit).
>
> Mystifyingly, in ~1% of datasets, one or the other route misses one or two
> indices.  Our work-around is to go belt-and-braces and run both for each
> dataset.
>
>
> It does however remain a bug.  Does anybody have any idea what's
> happening?  We can send example datasets to any volunteers who want to
> fiddle with it.
>
> phx
>
>
>

Re: [ccp4bb] (arcane) How to generate complete set of indices at low res

[Pearce, N.M. (Nick)]

Could you expand a bit on what you mean by a “putative” systematic absence? 
(e.g. why only the lowest order hkl?)



On 5 Apr 2018, at 19:39, James Holton 
> wrote:


You need to be careful with the exact space group at the particular stage in 
your pipeline here.  Often the lowest-order hkl is a putative systematic 
absence, so if you uniqueify in P222 you will get it, but if you uniqueify in 
P212121, then you won't.  That sort of thing.  Note that it doesn't matter what 
the "true" space group is, it only matters what is in the mtz header when you 
run uniqueify.

Could that be what is going on?

-James Holton

MAD Scisntist

On 4/5/2018 3:52 AM, Frank von Delft wrote:

Hello - can anybody shed light on this mystery:

We need (for PanDDA analysis) a lot of datasets each to have the complete set 
of low resolution indices, whether measured or not.  (Refmac adds the estimates 
as DFc, which is crucial when comparing maps.)

In ccp4, there are two obvious ways to get these indices complete:

  *   uniqueify
  *   CAD using the keyword "RESOLUTION FILE 1 999 "  (999 is the low 
resolution limit).

Mystifyingly, in ~1% of datasets, one or the other route misses one or two 
indices.  Our work-around is to go belt-and-braces and run both for each 
dataset.


It does however remain a bug.  Does anybody have any idea what's happening?  We 
can send example datasets to any volunteers who want to fiddle with it.

phx

Re: [ccp4bb] (arcane) How to generate complete set of indices at low res

[James Holton]

You need to be careful with the exact space group at the particular 
stage in your pipeline here.  Often the lowest-order hkl is a putative 
systematic absence, so if you uniqueify in P222 you will get it, but if 
you uniqueify in P212121, then you won't. That sort of thing.  Note that 
it doesn't matter what the "true" space group is, it only matters what 
is in the mtz header when you run uniqueify.


Could that be what is going on?

-James Holton

MAD Scisntist


On 4/5/2018 3:52 AM, Frank von Delft wrote:


Hello - can anybody shed light on this mystery:

We need (for PanDDA analysis) a lot of datasets each to have the 
complete set of low resolution indices, whether measured or not.  
(Refmac adds the estimates as DFc, which is crucial when comparing maps.)


In ccp4, there are two obvious ways to get these indices complete:

  * uniqueify
  * CAD using the keyword "RESOLUTION FILE 1 999 "  (999 is
the low resolution limit).

Mystifyingly, in ~1% of datasets, one or the other route misses one or 
two indices.  Our work-around is to go belt-and-braces and run both 
for each dataset.



It does however remain a bug.  Does anybody have any idea what's 
happening?  We can send example datasets to any volunteers who want to 
fiddle with it.


phx

Re: [ccp4bb] B-factor standardization

[John R Helliwell]

Dear Oliviero,
On one aspect of your query, this analysis dissected the different sources
of disorder contributions to B factors:-
Diamond, R Acta Cryst (1990). *A46*, 425-435
All best wishes,
John


On Thu, Apr 5, 2018 at 2:49 PM, Oliviero Carugo <
oliviero.car...@univie.ac.at> wrote:

> Dears,
>
> everybody knows that B-factors may change amongst different crystal
> structures and that they need to be standardized when different protein
> crystal structures are compared.
>
> If I am not wrong, I remember that someone proposed to standardize
> B-factors of protein atoms as “BS = B - Bave”, where Bave is the average
> B-factor of the protein. Such standardization is based on the hypothesis
> that independent sources of disorder add in determining the final B-factor.
> BS should represent atomic B-factors depurated by all factors different
> from atom oscillation, since Bave differences are neutralized.
>
> Does anyone can help me in finding a publication (80s or 90s) where these
> BS values are used?
>
> Thanks!
>
> Oliviero
>



-- 
Professor John R Helliwell DSc

Re: [ccp4bb] B-factor standardization

[Ethan Merritt]

On Thursday, 05 April 2018 15:49:44 Oliviero Carugo wrote:
> Dears,
> 
> everybody knows that B-factors may change amongst different crystal 
> structures and that they need to be standardized when different protein 
> crystal structures are compared.
> 
> If I am not wrong, I remember that someone proposed to standardize 
> B-factors of protein atoms as “BS = B - Bave”, where Bave is the average 
> B-factor of the protein. Such standardization is based on the hypothesis 
> that independent sources of disorder add in determining the final 
> B-factor. BS should represent atomic B-factors depurated by all factors 
> different from atom oscillation, since Bave differences are neutralized.

That sounds like flawed logic to me.
If you were going to do this at all, I think you would have to 
start by comparing the residual B factors after removing TLS
contributions.

> Does anyone can help me in finding a publication (80s or 90s) where 
> these BS values are used?

I think there are better tools available now than there were 
25 years ago.  I wouldn't go back that far to choose one without
first considering the work and thought that has been put into
structure analysis in the meantime.  If nothing else, you might
consider that "Bave" as calculated from the "Biso" (really Beq)
column in typical PDB files is a less than perfect approximation
[Acta Cryst. 2012 A67:512-516]

Ethan

> Thanks!
> 
> Oliviero

-- 
Ethan A Merritt, Dept of Biochemistry
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742

Re: [ccp4bb] (arcane) How to generate complete set of indices at low res

[Kay Diederichs]

Hi Frank,

could you please be more specific, and give examples - the more the better? 
That would help enormously in debugging. Information required is cell, 
spacegroup, and which reflection(s) (as h,k,l triples) is/are missing. 

best,

Kay


On Thu, 5 Apr 2018 11:52:37 +0100, Frank von Delft 
 wrote:

>Hello - can anybody shed light on this mystery:
>
>We need (for PanDDA analysis) a lot of datasets each to have the 
>complete set of low resolution indices, whether measured or not. (Refmac 
>adds the estimates as DFc, which is crucial when comparing maps.)
>
>In ccp4, there are two obvious ways to get these indices complete:
>
>  * uniqueify
>  * CAD using the keyword "RESOLUTION FILE 1 999 " (999 is the
>low resolution limit).
>
>Mystifyingly, in ~1% of datasets, one or the other route misses one or 
>two indices.  Our work-around is to go belt-and-braces and run both for 
>each dataset.
>
>
>It does however remain a bug.  Does anybody have any idea what's 
>happening?  We can send example datasets to any volunteers who want to 
>fiddle with it.
>
>phx
>
>
>

Re: [ccp4bb] B-factor standardization

[Daniel M. Himmel, Ph. D.]

Oliviero,
We published a paper in 2003 in which we normalized B-factors
to do a comparison of relative mobility or flexibility.  The reference
is:  Gourinath et al.  Structure 11:1621-1627 (2003).  The jargon
we used for Bave of the protein is .  In our case, to be
conservative in our interpretation of B-factor comparisons, we
excluded random turns from the  calculation and only
included alpha helices and beta sheets, because we were trying
to get a handle on the degree of excess flexibility of the less
ordered regions of a large structure.

-Daniel


On Thu, Apr 5, 2018 at 9:49 AM, Oliviero Carugo <
oliviero.car...@univie.ac.at> wrote:

> Dears,
>
> everybody knows that B-factors may change amongst different crystal
> structures and that they need to be standardized when different protein
> crystal structures are compared.
>
> If I am not wrong, I remember that someone proposed to standardize
> B-factors of protein atoms as “BS = B - Bave”, where Bave is the average
> B-factor of the protein. Such standardization is based on the hypothesis
> that independent sources of disorder add in determining the final B-factor.
> BS should represent atomic B-factors depurated by all factors different
> from atom oscillation, since Bave differences are neutralized.
>
> Does anyone can help me in finding a publication (80s or 90s) where these
> BS values are used?
>
> Thanks!
>
> Oliviero
>

Re: [ccp4bb] (arcane) How to generate complete set of indices at low res

[Ian Clifton]

I  wrote:

> Frank von Delft  writes:
>
>> No, the point is: uniqueify manages to not always do this. 
>
> The “uniqueify” script depends on the “unique” program, which seems to
> contain a built‐in low‐resolution limit of 50Å. Could it be this isn’t
> always low enough?

No, scrub that—the low resolution limit of the file that comes out
depends on the cell that you put in (that’ll teach me to use round
numbers for my test cell).
-- 
Ian ◎

Re: [ccp4bb] (arcane) How to generate complete set of indices at low res

[Ian Clifton]

Frank von Delft  writes:

> No, the point is: uniqueify manages to not always do this. 

The “uniqueify” script depends on the “unique” program, which seems to
contain a built‐in low‐resolution limit of 50Å. Could it be this isn’t
always low enough?
-- 
Ian Clifton ⚗ ℡: +44 1865 275677
Chemistry Research Laboratory ℻: +44 1865 285002
Oxford University : ian.clif...@chem.ox.ac.uk
Mansfield Road   Oxford OX1 3TA   UK

[ccp4bb] B-factor standardization

[Oliviero Carugo]

Dears,

everybody knows that B-factors may change amongst different crystal 
structures and that they need to be standardized when different protein 
crystal structures are compared.


If I am not wrong, I remember that someone proposed to standardize 
B-factors of protein atoms as “BS = B - Bave”, where Bave is the average 
B-factor of the protein. Such standardization is based on the hypothesis 
that independent sources of disorder add in determining the final 
B-factor. BS should represent atomic B-factors depurated by all factors 
different from atom oscillation, since Bave differences are neutralized.


Does anyone can help me in finding a publication (80s or 90s) where 
these BS values are used?


Thanks!

Oliviero

Re: [ccp4bb] (arcane) How to generate complete set of indices at low res

[Frank von Delft]

No, the point is:  uniqueify manages to not /alw//ays/ do this.

I suppose I'm really asking:  who wants an example file to debug it?  
Because we have failed utterly.


Frank


On 05/04/2018 12:04, Eleanor Dodson wrote:

You need to be a bit more specific! - unbiqueify is meant to do this..
I didnt know CAD would generate indices not in the input file, unless 
you asked to generate a full set of FreeR terms, when the job I 
thought ran uniqueify?

Eleanor

On 5 April 2018 at 11:52, Frank von Delft > wrote:


Hello - can anybody shed light on this mystery:

We need (for PanDDA analysis) a lot of datasets each to have the
complete set of low resolution indices, whether measured or not. 
(Refmac adds the estimates as DFc, which is crucial when comparing
maps.)

In ccp4, there are two obvious ways to get these indices complete:

  * uniqueify
  * CAD using the keyword "RESOLUTION FILE 1 999 "  (999
is the low resolution limit).

Mystifyingly, in ~1% of datasets, one or the other route misses
one or two indices.  Our work-around is to go belt-and-braces and
run both for each dataset.


It does however remain a bug.  Does anybody have any idea what's
happening?  We can send example datasets to any volunteers who
want to fiddle with it.

phx

Re: [ccp4bb] (arcane) How to generate complete set of indices at low res

[Eleanor Dodson]

You need to be a bit more specific! - unbiqueify is meant to do this..
I didnt know CAD would generate indices not in the input file, unless you
asked to generate a full set of FreeR terms, when the job I thought ran
uniqueify?
Eleanor

On 5 April 2018 at 11:52, Frank von Delft 
wrote:

> Hello - can anybody shed light on this mystery:
>
> We need (for PanDDA analysis) a lot of datasets each to have the complete
> set of low resolution indices, whether measured or not.  (Refmac adds the
> estimates as DFc, which is crucial when comparing maps.)
>
> In ccp4, there are two obvious ways to get these indices complete:
>
>- uniqueify
>- CAD using the keyword "RESOLUTION FILE 1 999 "  (999 is the
>low resolution limit).
>
> Mystifyingly, in ~1% of datasets, one or the other route misses one or two
> indices.  Our work-around is to go belt-and-braces and run both for each
> dataset.
>
>
> It does however remain a bug.  Does anybody have any idea what's
> happening?  We can send example datasets to any volunteers who want to
> fiddle with it.
>
> phx
>
>
>

[ccp4bb] (arcane) How to generate complete set of indices at low res

[Frank von Delft]

Hello - can anybody shed light on this mystery:

We need (for PanDDA analysis) a lot of datasets each to have the 
complete set of low resolution indices, whether measured or not. (Refmac 
adds the estimates as DFc, which is crucial when comparing maps.)


In ccp4, there are two obvious ways to get these indices complete:

 * uniqueify
 * CAD using the keyword "RESOLUTION FILE 1 999 " (999 is the
   low resolution limit).

Mystifyingly, in ~1% of datasets, one or the other route misses one or 
two indices.  Our work-around is to go belt-and-braces and run both for 
each dataset.



It does however remain a bug.  Does anybody have any idea what's 
happening?  We can send example datasets to any volunteers who want to 
fiddle with it.


phx