[ccp4bb] Newly funded Postdoctoral position — Rogala Lab @ Stanford

[Kacper Rogala]

Dear CCP4 Community,

My lab at Stanford (https://rogala.stanford.edu) is thrilled to announce a
newly funded position for a postdoctoral scholar with a background in
structural biology or protein biochemistry. Expertise in working with
either integral or peripheral membrane proteins will be an advantage, but
it is not a requirement. Compensation of our postdocs is set to the
Stanford rates .

Our lab is relatively new, and we are in search of driven candidates with a
start-up mentality and a deep interest in deciphering protein mechanisms.
Please share this opportunity with your soon-to-graduate PhD students who
may be looking for their next career step! We have a flexible start date,
so if you anticipate that your PhD might take another year to complete but
you like our research, we encourage you to apply.

Please see the official ad on the Stanford postdoc website here
,
and a short blurb below — describing what our lab is about.

Potential candidates, please write a few lines about yourself — about your
previous research experience, and what you liked about our lab that got you
interested! Please include your CV and contact details to three references.

Many thanks and best wishes,

Kacper

KACPER ROGALA, D.PHIL.

*Assistant Professor*

*–––-*

Department of Structural Biology


Department of Chemical and Systems Biology

Stanford Cancer Institute 

Stanford University School of Medicine

Biomedical Innovations Building
240 Pasteur Drive, 4700 | Palo Alto, CA, 94304

https://rogala.stanford.edu

We are a team of structural and chemical biologists fascinated by how cells
control their metabolism in response to nutrients. How are nutrients
recognized by their protein sensors? How is their transport across cellular
and intracellular membranes regulated? And, how is nutrient sensing
integrated with other chemical signals, such as growth factors, to
determine cellular decisions, especially the decision: to grow or not to
grow? We are aiming to answer these fundamental questions at the level of
ångstroms, nanometers, and micrometers — with cryo-EM, X-ray
crystallography, and a full range of other techniques. Many proteins in
nutrient signaling pathways are deregulated in cancer, and in parallel to
the mechanistic structural work, we are also developing targeted chemical
probes to modulate activity of these proteins in cells and organisms.

We primarily work on proteins that associate with biological membranes —
either as large peripheral membrane complexes, or as integral membrane
transporters.

Our latest papers on this topic are:

   -

   (REVIEW) Linde-Garelli and Rogala (2023) Structural mechanisms of the
   mTOR pathway. Current Opinion in Structural Biology. 82:102663. PMID:
   37572585 .
   -

   Valenstein and Rogala et al. (2022) Structure of the nutrient-sensing
   hub GATOR2. Nature, 607(7919):610-616. PMID: 35831510
   .
   -

   Rogala et al. (2019) Structural basis for the docking of mTORC1 on the
   lysosomal surface. Science, 366(6464):468-475. PMID: 31601708
   .
   -

   Shen and Rogala et al. (2019) Cryo-EM structure of the human
   FLCN-FNIP2-Rag-Ragulator complex. Cell, 179(6):1319-1329.e8. PMID:
   31704029 .



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Re: [ccp4bb] Occupancy Refinement limitation

[Eleanor Dodson]

Low tech but simple.
Exclude both conformers from the refinement. (set occs to 0.00 so you still
see the coordinates in COOT)
Look at the difference map density and make a rough estimate of occupancy
from peak heights for a "fixed" bit of the conformers..

The relative ratios for different states should show up...
Eleanor

On Wed, 6 Sept 2023 at 11:49,  wrote:

> I would be tempted to try phenix.ensemble_refinement instead.
>
> Look at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3949522/
> or
> https://phenix-online.org/phenixwebsite_static/mainsite/files/presentations/ensemble_refinement_burnley_at_al_10DEC2012.pdf
>
> or https://elifesciences.org/articles/00311
>
> Tassos
>
> On 6 Sep 2023, at 12:37, Matt Mcleod  wrote:
>
> Hi,
>
> I should be a bit more specific.
>
> We have many crystal structures to indicate that a loop adopts
> conformation A and conformation B, or - the loop can be disordered where
> the electron density is washed out.  These states are dependent on how we
> perturb the system.
>
> We have a series of data, as a function of osmolyte concentration, that
> adjusts this equilibrium and we want to put a numerical value onto it.
> Resolution is between 1.7 - 2.0 A. This is non-anomalous data but certainly
> would be helpful in the future to include if possible some anomalous
> scatters. Have been using phenix.refine.
>
> So what we have done is modeled both conformation A and B and are refining
> the data in order to get the occupancies (fixing B-factors) regardless of
> if there is electron density present for one of the two conformations since
> in some cases (likely all) there will be a population of both A, B, and
> disordered and the relative true occupancies will move.  I am trying to
> sort out how accurate these occupancy values are as opposed to showing the
> electron density for each conformation fit at some common threshold.  My
> general sense is that if it is modeled but no electron density, there will
> be a non-zero value and vice versa if it is the only conformation present
> it will be less than unity.
>
> I will take a look at the references!  Very much appreciated.
> Matt
>
> On Wed, 6 Sept 2023 at 02:08, Eleanor Dodson <
> 176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:
>
>> Well - occupancy refinement is particularly imprecise, and highly
>> correlated with temperature factors.
>> Also the population for a surface ARG or LYS may well have more than two
>> conformations, whereas some internal residue is better defined.
>> There is also the Q of solvent - dual occupancies will generate dual
>> solvent networks..
>>
>> ..You dont say what resolution your data are. Again at 0.8A you can be
>> confident - at 3A it is at best a guess. So I for one do not take the
>> numbers very seriously - they are a flag only.
>>
>> Presumably you didnt model the second conformation unless there was some
>> feature in an earlier map to suggest it existed?
>> Good luck Eleanor
>>
>>
>> On Wed, 6 Sept 2023 at 03:18, Pavel Afonine  wrote:
>>
>>> Hi Matt,
>>> I believe figure 3 here:
>>> https://www.nature.com/articles/s41467-018-06957-w
>>> is relevant to your question.
>>> Pavel
>>>
>>>
>>> On Tue, Sep 5, 2023 at 11:32 AM Matt McLeod 
>>> wrote:
>>>
 Hi all,

 I am trying to get some insight in the accuracy/precision of occupancy
 refinements.  I have done some 2-state occupancy refinements and have
 observed the refinement achieving ~0.25-0.3 occupancy for the minor
 population.  This population, when observing the electron density maps, had
 essentially no evidence for it being present.  I was wondering:

 What are the errors in the reported occupancies?

 Is there a lower and upper limit to occupancy refinements?  As in, if
 you occupancy refine two states and one is imaginary will it refine to
 approximately 1 and 0?  Or does the background noise always given a
 positive number to the imaginary set?  This would, to me at least, be the
 lower and upper limits to the occupancy refinements and could be used as a
 normalization factor for other atoms.  Maybe my logic is off...

 Any insight or literature would be appreciated!
 Matt

 

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Re: [ccp4bb] Density at a short distance near Tyrosine

[Gerard Bricogne]

Dear Lumbini,

 Thank you for sharing this picture.

 Considering the low resolution and modest quality of your map, as
manifested in the "blobbiness" of the side chains near the green peak, the
"short distance" you mention is perhaps quite imprecise and thus not such a
barrier to tentatively placing a water molecule in that peak. What happens
if you do and re-refine?


 Best wishes,

Gerard

--
On Wed, Sep 06, 2023 at 06:42:03PM +0530, Lumbini Yadav wrote:
> Dear Matthew,
> Below is the jpeg attached  for the density at 4sigma.
> 
> On Wed, Sep 6, 2023 at 6:21 PM Matthew Snee 
> wrote:
> 
> > Is the density big enough for it to be phospho-tyrosine?
> >
> > Best wishes
> >
> > Matthew.
> > --
> > *From:* CCP4 bulletin board  on behalf of Lumbini
> > Yadav 
> > *Sent:* 06 September 2023 13:47
> > *To:* CCP4BB@JISCMAIL.AC.UK 
> > *Subject:* [ccp4bb] Density at a short distance near Tyrosine
> >
> > Hi all, I have determined the structure of a protein at 3Å resolution. The
> > protein has 6 chains in the asymmetric unit. In all these 6 chains I see a
> > density near tyrosine which is at a distance of 2. 1 from the OH group of
> > tyrosine. Since this
> > ZjQcmQRYFpfptBannerStart
> > This Message Is From a New External Sender
> > You have not previously corresponded with this sender. Please exercise
> > caution when opening links or attachments included in this message.
> >
> > ZjQcmQRYFpfptBannerEnd
> >
> > Hi all,
> >
> > I have determined the structure of a protein at 3Å resolution. The
> > protein has 6 chains in the asymmetric unit. In all these 6 chains I see a
> > density near tyrosine which is at a distance of 2.1 from the OH group of
> > tyrosine. Since this distance is short for a hydrogen bond we are skeptical
> > about adding water molecules at this position.  We have ruled out the
> > presence of a metal ion at that position.
> >
> > Any insight on what should be added in this density at this short distance
> > would be appreciated
> >
> > Thank you
> >
> >
> >
> > Regards
> >
> > Lumbini
> >
> > --
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
> > [jiscmail.ac.uk]
> > 
> >
> 
> 
> 
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[ccp4bb] Density at a short distance near Tyrosine

[Lumbini Yadav]

Hi all,

I have determined the structure of a protein at 3Å resolution. The protein
has 6 chains in the asymmetric unit. In all these 6 chains I see a density
near tyrosine which is at a distance of 2.1 from the OH group of tyrosine.
Since this distance is short for a hydrogen bond we are skeptical about
adding water molecules at this position.  We have ruled out the presence of
a metal ion at that position.

Any insight on what should be added in this density at this short distance
would be appreciated

Thank you



Regards

Lumbini



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Re: [ccp4bb] Occupancy Refinement limitation

[a . perrakis]

I would be tempted to try phenix.ensemble_refinement instead.

Look at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3949522/
or 
https://phenix-online.org/phenixwebsite_static/mainsite/files/presentations/ensemble_refinement_burnley_at_al_10DEC2012.pdf
or https://elifesciences.org/articles/00311

Tassos

On 6 Sep 2023, at 12:37, Matt Mcleod 
mailto:mjmcleo...@gmail.com>> wrote:

Hi,

I should be a bit more specific.

We have many crystal structures to indicate that a loop adopts conformation A 
and conformation B, or - the loop can be disordered where the electron density 
is washed out.  These states are dependent on how we perturb the system.

We have a series of data, as a function of osmolyte concentration, that adjusts 
this equilibrium and we want to put a numerical value onto it.  Resolution is 
between 1.7 - 2.0 A. This is non-anomalous data but certainly would be helpful 
in the future to include if possible some anomalous scatters. Have been using 
phenix.refine.

So what we have done is modeled both conformation A and B and are refining the 
data in order to get the occupancies (fixing B-factors) regardless of if there 
is electron density present for one of the two conformations since in some 
cases (likely all) there will be a population of both A, B, and disordered and 
the relative true occupancies will move.  I am trying to sort out how accurate 
these occupancy values are as opposed to showing the electron density for each 
conformation fit at some common threshold.  My general sense is that if it is 
modeled but no electron density, there will be a non-zero value and vice versa 
if it is the only conformation present it will be less than unity.

I will take a look at the references!  Very much appreciated.
Matt

On Wed, 6 Sept 2023 at 02:08, Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
 wrote:
Well - occupancy refinement is particularly imprecise, and highly correlated 
with temperature factors.
Also the population for a surface ARG or LYS may well have more than two 
conformations, whereas some internal residue is better defined.
There is also the Q of solvent - dual occupancies will generate dual solvent 
networks..

..You dont say what resolution your data are. Again at 0.8A you can be 
confident - at 3A it is at best a guess. So I for one do not take the numbers 
very seriously - they are a flag only.

Presumably you didnt model the second conformation unless there was some 
feature in an earlier map to suggest it existed?
Good luck Eleanor


On Wed, 6 Sept 2023 at 03:18, Pavel Afonine 
mailto:pafon...@gmail.com>> wrote:
Hi Matt,
I believe figure 3 here:
https://www.nature.com/articles/s41467-018-06957-w
is relevant to your question.
Pavel


On Tue, Sep 5, 2023 at 11:32 AM Matt McLeod 
mailto:mjmcleo...@gmail.com>> wrote:
Hi all,

I am trying to get some insight in the accuracy/precision of occupancy 
refinements.  I have done some 2-state occupancy refinements and have observed 
the refinement achieving ~0.25-0.3 occupancy for the minor population.  This 
population, when observing the electron density maps, had essentially no 
evidence for it being present.  I was wondering:

What are the errors in the reported occupancies?

Is there a lower and upper limit to occupancy refinements?  As in, if you 
occupancy refine two states and one is imaginary will it refine to 
approximately 1 and 0?  Or does the background noise always given a positive 
number to the imaginary set?  This would, to me at least, be the lower and 
upper limits to the occupancy refinements and could be used as a normalization 
factor for other atoms.  Maybe my logic is off...

Any insight or literature would be appreciated!
Matt



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--
Matthew Jordan McLeod, PhD
Post-Doctoral Fellow - Cornell University






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This m

Re: [ccp4bb] Occupancy Refinement limitation

[Matt Mcleod]

Hi,

I should be a bit more specific.

We have many crystal structures to indicate that a loop adopts conformation
A and conformation B, or - the loop can be disordered where the electron
density is washed out.  These states are dependent on how we perturb the
system.

We have a series of data, as a function of osmolyte concentration, that
adjusts this equilibrium and we want to put a numerical value onto it.
Resolution is between 1.7 - 2.0 A. This is non-anomalous data but certainly
would be helpful in the future to include if possible some anomalous
scatters. Have been using phenix.refine.

So what we have done is modeled both conformation A and B and are refining
the data in order to get the occupancies (fixing B-factors) regardless of
if there is electron density present for one of the two conformations since
in some cases (likely all) there will be a population of both A, B, and
disordered and the relative true occupancies will move.  I am trying to
sort out how accurate these occupancy values are as opposed to showing the
electron density for each conformation fit at some common threshold.  My
general sense is that if it is modeled but no electron density, there will
be a non-zero value and vice versa if it is the only conformation present
it will be less than unity.

I will take a look at the references!  Very much appreciated.
Matt

On Wed, 6 Sept 2023 at 02:08, Eleanor Dodson <
176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

> Well - occupancy refinement is particularly imprecise, and highly
> correlated with temperature factors.
> Also the population for a surface ARG or LYS may well have more than two
> conformations, whereas some internal residue is better defined.
> There is also the Q of solvent - dual occupancies will generate dual
> solvent networks..
>
> ..You dont say what resolution your data are. Again at 0.8A you can be
> confident - at 3A it is at best a guess. So I for one do not take the
> numbers very seriously - they are a flag only.
>
> Presumably you didnt model the second conformation unless there was some
> feature in an earlier map to suggest it existed?
> Good luck Eleanor
>
>
> On Wed, 6 Sept 2023 at 03:18, Pavel Afonine  wrote:
>
>> Hi Matt,
>> I believe figure 3 here:
>> https://www.nature.com/articles/s41467-018-06957-w
>> is relevant to your question.
>> Pavel
>>
>>
>> On Tue, Sep 5, 2023 at 11:32 AM Matt McLeod  wrote:
>>
>>> Hi all,
>>>
>>> I am trying to get some insight in the accuracy/precision of occupancy
>>> refinements.  I have done some 2-state occupancy refinements and have
>>> observed the refinement achieving ~0.25-0.3 occupancy for the minor
>>> population.  This population, when observing the electron density maps, had
>>> essentially no evidence for it being present.  I was wondering:
>>>
>>> What are the errors in the reported occupancies?
>>>
>>> Is there a lower and upper limit to occupancy refinements?  As in, if
>>> you occupancy refine two states and one is imaginary will it refine to
>>> approximately 1 and 0?  Or does the background noise always given a
>>> positive number to the imaginary set?  This would, to me at least, be the
>>> lower and upper limits to the occupancy refinements and could be used as a
>>> normalization factor for other atoms.  Maybe my logic is off...
>>>
>>> Any insight or literature would be appreciated!
>>> Matt
>>>
>>> 
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>>>
>>> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
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>>
>> --
>>
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>
> --
>
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-- 
*Matthew Jordan McLeod, PhD*
*Post-Doctoral Fellow - Cornell University*



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Re: [ccp4bb] Occupancy Refinement limitation

[John R Helliwell]

Dear Matt,I believe this study we made may well assist your evaluations:-(IUCr) Experience with exchange and archiving of raw data: comparison of data from two diffractometers and four software packages on a series of lysozyme crystalsjournals.iucr.orgBest wishes,John Emeritus Professor John R Helliwell DScOn 5 Sep 2023, at 19:32, Matt McLeod  wrote:Hi all,I am trying to get some insight in the accuracy/precision of occupancy refinements.  I have done some 2-state occupancy refinements and have observed the refinement achieving ~0.25-0.3 occupancy for the minor population.  This population, when observing the electron density maps, had essentially no evidence for it being present.  I was wondering:What are the errors in the reported occupancies?Is there a lower and upper limit to occupancy refinements?  As in, if you occupancy refine two states and one is imaginary will it refine to approximately 1 and 0?  Or does the background noise always given a positive number to the imaginary set?  This would, to me at least, be the lower and upper limits to the occupancy refinements and could be used as a normalization factor for other atoms.  Maybe my logic is off...Any insight or literature would be appreciated!MattTo unsubscribe from the CCP4BB list, click the following link:https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/

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Re: [ccp4bb] Occupancy Refinement limitation

[Tim Gruene]

Hi Matt,

this can be done with SHELXL, even at 2-3A resolution. Anomalous data
do help reduce the correlation between B-factor and occupancy 
(see e.g. https://doi.org/10.1126/science.1254840 to stick with Pavel's
example)

SHELXL prints the correlation coefficient between occupancy and
b-factor, and also the ESD of the occupancy. The is more reliable with
least-square refinement, but for large structures, you may need a lot
of RAM.

PDB2INS (https://www.nature.com/articles/s41467-018-06957-w)
conveniently prepares the INS-file from your PDB for you.

Best wishes,
Tim

On Tue, 5 Sep 2023 19:31:25 +0100 Matt McLeod 
wrote:

> Hi all,
> 
> I am trying to get some insight in the accuracy/precision of
> occupancy refinements.  I have done some 2-state occupancy
> refinements and have observed the refinement achieving ~0.25-0.3
> occupancy for the minor population.  This population, when observing
> the electron density maps, had essentially no evidence for it being
> present.  I was wondering:
> 
> What are the errors in the reported occupancies?
> 
> Is there a lower and upper limit to occupancy refinements?  As in, if
> you occupancy refine two states and one is imaginary will it refine
> to approximately 1 and 0?  Or does the background noise always given
> a positive number to the imaginary set?  This would, to me at least,
> be the lower and upper limits to the occupancy refinements and could
> be used as a normalization factor for other atoms.  Maybe my logic is
> off...
> 
> Any insight or literature would be appreciated!
> Matt
> 
> 
> 
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-- 
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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Description: OpenPGP digital signature