Re: [ccp4bb] REGISTRATION OPEN: Engineering Recombinant Proteins: London, Nov 4-5

[Amir Khan]

Hi

This is a reminder that the 'early-bird’ registration deadline (4 Sep) is 
approaching:
https://www.eventsforce.net/biochemsoc/frontend/reg/thome.csp?pageID=31653=71
 
<https://www.eventsforce.net/biochemsoc/frontend/reg/thome.csp?pageID=31653=71>
There will also be opportunities for attendees to bring protein samples for 
analyses by equipment suppliers.

Best,
Amir 


> 
> 
> 
>> On Jul 9, 2019, at 9:46 AM, Amir Khan > <mailto:k...@crystal.harvard.edu>> wrote:
>> 
>> Hi,
>> 
>> This is a reminder and additional details of an upcoming ‘Training Day’ 
>> organized by the
>> Biochemical Society.  
>> 
>> Engineering recombinant proteins for structural and functional studies
>> Tentative venue:  CIWEM, Saffron Hill, Holborn, London  EC1N 8QS (near 
>> Farringdon station)
>> Currently, fees are Member £120 / Non member £175 / Student £60, although we 
>> are working
>> to reduce the costs.
>> 
>> The programme will address the limiting step in structural studies - the 
>> production of 
>> pure and homogenous proteins.  Talks and discussions at this event will 
>> explore strategies 
>> to optimize membrane and soluble proteins for crystallization, cryo-EM and 
>> NMR studies.  
>> Discussions will include construct optimization, novel thermostability 
>> assays, 
>> crystallization tools and ‘tricks of the trade’ to enable structure 
>> determination. 
>> 
>> The list of invited speakers are experienced in the structure determination 
>> of 
>> membrane proteins, cytosolic/secreted proteins, and macromolecular signaling 
>> complexes: 
>> 
>> Edmund Kunji, Cambridge
>> Daniel Panne, Leicester
>> Maria Sasi Conte, King’s College
>> Naomi Chayen, Imperal College
>> Laura Itzhaki, Cambridge
>> Chris Tate, Cambridge
>> 
>> More details about the programme and venue will be provided in the coming 
>> weeks.
>> 
>> Best wishes,
>> 
>> Amir Khan, Harvard Medical School
>> Rivka Isaacson, King’s College London
>> 
> 
> 
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Re: [ccp4bb] Importance of temperature during initial crystallization screening

[Amir Khan]

Hate to self-promote, but these crystals were interesting, At 30deg, two 
crystal forms co-existed with
inverted solubility - one form melted when cooled, the other melted upon 
heating.

https://www.sciencedirect.com/science/article/abs/pii/S0006349519305879 




> On Aug 2, 2019, at 10:15 AM, Jared Sampson  wrote:
> 
> Dear all -
> 
> I have also had success with crystallization above room temperature.  I once 
> grew diffracting crystals of a small, thermostable enzyme by incubating the 
> plates in a 37°C bacterial culture incubator, and despite similar reservoir 
> conditions, these high-temperature crystals grew in a different crystal form 
> from those of the same enzyme at room temperature.  To harvest and freeze, I 
> opted to keep the plate warm in case I wanted to go back to it later (and to 
> prevent condensation on the cover slips), so I set up a small humidifier next 
> to the stereo microscope, mounted a heat gun across the bench set to the 
> lowest setting, and adjusted the distance until a thermometer mounted near 
> the microscope stage indicated a stable 37°C.  It was low-tech, but it worked 
> well enough.
> 
> Cheers,
> Jared
> 
> 
> 
> On August 2, 2019 at 4:48:06 AM, Pierre Rizkallah (rizkall...@cardiff.ac.uk 
> ) wrote:
> 
>> Hi Everyone,
>> 
>>  
>> 
>> Look up J Mol Biol 270 724-738 (Valegard et al.), or doi 
>> https://doi.org/10.1006/jmbi.1997.1144 
>>  , PDB entry 1ZDH, where the M 
>> section describes crystallising MS2 virus at 37C. From memory, as they were 
>> collecting data at Daresbury during my time there, the crystals were in a 
>> gel, after bringing the set up to RT. It helped them a great deal, because 
>> then they could cut out the gel and orient the crystals favourably from 
>> knowledge of the morphology. That was way before cryo-cooling was available, 
>> and a complete data set collection used to require lots of crystals. It 
>> helped to know where the gaps in the rotation range were.
>> 
>>  
>> 
>> Although I haven’t done any experiment at a temp higher than RT, I expect 
>> that if you get crystals at a higher temp, then you would likely have them 
>> better ordered as you ‘cool’ them down to RT. There is an added advantage in 
>> creature comfort without compromising the precious little crystals. Win-win?
>> 
>>  
>> 
>> Pierre
>> 
>> ***
>> 
>> Dr Pierre Rizkallah, Senior Lecturer Structural Biology
>> 
>> Institute of Infection & Immunology, Sir Geraint Evans Building, 
>> 
>> School of Medicine, Heath Campus, Cardiff, CF14 4XN
>> 
>> email: rizkall...@cardiff.ac.uk 
>> phone: +44 29 2074 2248
>> 
>> http://www.cardiff.ac.uk/people/view/126690-rizkallah-pierre 
>> 
>>  
>> 
>> From: CCP4 bulletin board > > On Behalf Of Newman, Janet (Manufacturing, 
>> Parkville)
>> Sent: 01 August 2019 23:30
>> To: CCP4BB@JISCMAIL.AC.UK 
>> Subject: Re: [ccp4bb] Importance of temperature during initial 
>> crystallization screening
>> 
>>  
>> 
>> Interesting topic,
>> 
>>  
>> 
>> Certainly the two papers suggested by Georg are relevant, and I fully agree 
>> with the comments from Daniel that it is hard to predict the behaviour of 
>> any given protein from a statistical analysis of proteins in general. 
>> 
>>  
>> 
>> I find it interesting that even with the use of incubators to store and 
>> image crystal experiments (which takes away the issue of setting up plates 
>> in the cold) we still find that our facility users have a strong bias 
>> towards 20C over 8C – for example, our standard initial screen (Shotgun) has 
>> been set up just over 360 times in the last year, 216 times at 20C and 135 
>> times at 8C. It is equally easy to type “20” or “8” into the request for the 
>> plate storage temperature, so its not ease of crystallisation setup which 
>> dictates this. It might well be that the thought of harvesting crystals from 
>> cold plates puts people off the lower temperature?
>> 
>>  
>> 
>> A quick search through our database shows that of the shotgun screens that 
>> were set up in the last year, 51% of those set up at 20C were (human) scored 
>> as containing crystals, and 47% of those set up at 8C were (human) scored as 
>> containing crystals. So the rate of crystal formation at the two temperature 
>> is essentially the same.
>> 
>> Patrick, I can’t comment on “unusual” temperatures, as I don’t have any 
>> significant experience with going outside the 4-20 region.
>> 
>>  
>> 
>> Cheers, Janet
>> 
>>  
>> 
>>  
>> 
>> From: CCP4 bulletin board > > On Behalf Of Georg Mlynek
>> Sent: Friday, 2 August 2019 5:09 AM
>> To: CCP4BB@JISCMAIL.AC.UK 

[ccp4bb] REGISTRATION OPEN: Engineering Recombinant Proteins: London, Nov 4-5

[Amir Khan]

Hi,

I’d like to advise you that registration is open, and the venue in London 
(CIWEM)
has been confirmed.  
I would advise registering early given the early responses that we have seen.

Kind regards,

Amir 


> On Jul 9, 2019, at 9:46 AM, Amir Khan  wrote:
> 
> Hi,
> 
> This is a reminder and additional details of an upcoming ‘Training Day’ 
> organized by the
> Biochemical Society.  
> 
> Engineering recombinant proteins for structural and functional studies
> Tentative venue:  CIWEM, Saffron Hill, Holborn, London  EC1N 8QS (near 
> Farringdon station)
> Currently, fees are Member £120 / Non member £175 / Student £60, although we 
> are working
> to reduce the costs.
> 
> The programme will address the limiting step in structural studies - the 
> production of 
> pure and homogenous proteins.  Talks and discussions at this event will 
> explore strategies 
> to optimize membrane and soluble proteins for crystallization, cryo-EM and 
> NMR studies.  
> Discussions will include construct optimization, novel thermostability 
> assays, 
> crystallization tools and ‘tricks of the trade’ to enable structure 
> determination. 
> 
> The list of invited speakers are experienced in the structure determination 
> of 
> membrane proteins, cytosolic/secreted proteins, and macromolecular signaling 
> complexes: 
> 
> Edmund Kunji, Cambridge
> Daniel Panne, Leicester
> Maria Sasi Conte, King’s College
> Naomi Chayen, Imperal College
> Laura Itzhaki, Cambridge
> Chris Tate, Cambridge
> 
> More details about the programme and venue will be provided in the coming 
> weeks.
> 
> Best wishes,
> 
> Amir Khan, Harvard Medical School
> Rivka Isaacson, King’s College London
> 




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[ccp4bb] Engineering Recombinant Proteins: Bioch Soc Training day, London, Nov 4-5

[Amir Khan]

Hi,

This is a reminder and additional details of an upcoming ‘Training Day’ 
organized by the
Biochemical Society.  

Engineering recombinant proteins for structural and functional studies
Tentative venue:  CIWEM, Saffron Hill, Holborn, London  EC1N 8QS (near 
Farringdon station)
Currently, fees are Member £120 / Non member £175 / Student £60, although we 
are working
to reduce the costs.

The programme will address the limiting step in structural studies - the 
production of 
pure and homogenous proteins.  Talks and discussions at this event will explore 
strategies 
to optimize membrane and soluble proteins for crystallization, cryo-EM and NMR 
studies.  
Discussions will include construct optimization, novel thermostability assays, 
crystallization tools and ‘tricks of the trade’ to enable structure 
determination. 

The list of invited speakers are experienced in the structure determination of 
membrane proteins, cytosolic/secreted proteins, and macromolecular signaling 
complexes: 

Edmund Kunji, Cambridge
Daniel Panne, Leicester
Maria Sasi Conte, King’s College
Naomi Chayen, Imperal College
Laura Itzhaki, Cambridge
Chris Tate, Cambridge

More details about the programme and venue will be provided in the coming weeks.

Best wishes,

Amir Khan, Harvard Medical School
Rivka Isaacson, King’s College London


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[ccp4bb] Engineering Recombinant Proteins: Biochem Soc Training day, Nov 4-5, 2019

[Amir Khan]

Hi,

This is a ‘save-the-date’ message for an upcoming ‘Training Day’ organized by 
the
Biochemical Society.  The event will be held in London (UK) and is entitled:

‘Engineering recombinant proteins for structural and functional studies’

The programme will address the limiting step in structural studies - the 
production of
pure and homogenous proteins.  Talks and discussions at this event will explore 
strategies
to optimize membrane and soluble proteins for crystallization, cryo-EM and NMR 
studies.
Discussions will include construct optimization, novel thermostability assays,
crystallization tools and ‘tricks of the trade’ to enable structure 
determination.

This event is particlarly directed toward early-stage graduate students in 
structural biology.
However, anyone interested in generating soluble proteins for a variety of 
applications,
such as enzymatic assays and antibody production, would be welcome to attend.
Manufacturers of instruments and techniques to assay purity and homogeneity
will be available for ‘hands-on’ demonstrations.

The list of invited speakers are experienced in the structure determination of
membrane proteins, cytosolic/secreted proteins, and macromolecular signaling 
complexes:

Edmund Kunji, Cambridge
Daniel Panne, Leicester
Maria Sasi Conte, King’s College
Naomi Chayen, Imperal College
Laura Itzhaki, Cambridge
Chris Tate, Cambridge

More details about the programme and venue will be provided in the coming weeks.

Best wishes,

Amir Khan, Trinity College Dublin
Rivka Isaacson, King’s College




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[ccp4bb] average B-factors

[Amir Khan]

Dear all,

Sorry for naive CCP4 question, is there a simple way to calculate average 
B-factors (side chain, backbone, all)
for only part of a model, such as a domain, loop, etc…

Thanks,
Amir

[ccp4bb] PhD position at Trinity College Dublin

[Amir Khan]

Applications for a PhD position are invited for the protein X-ray 
crystallography group at Trinity College Dublin, Ireland.  The project involves 
a structural investigation of a viral protein in complex with a newly 
characterized sensor of the innate immune system.  More information and a 
publication list can be found on the web pages below.  The position is open to 
EU citizens and is fully funded (stipend + fees) for 3 years by Science 
Foundation Ireland, beginning in September, 2014.
The lab is located in the center of Dublin, which is a multi-cultural and 
vibrant city that is well connected to Europe and the USA.
Please email a cover letter, CV, and contact information for three references 
to:
Amir Khan
School of Biochemistry and Immunology
Trinity College Dublin
amir.k...@tcd.iemailto:amir.k...@tcd.ie
http://www.tcd.ie/Biochemistry/research/a_khan/index.php

[ccp4bb] positions open in Dublin, Ireland

[Amir Khan]

Applications from potential PhD students, post-doctoral fellows, and research 
technicians are invited for the protein X-ray crystallography group at Trinity 
College Dublin, Ireland.  The projects are diverse and involve the 
crystallization of bacterial/cellular protein complexes associated with 
membrane trafficking, as well as viral/cellular complexes that antagonize 
innate immunity.  More information and a publication list can be found on the 
web pages below.  Proficiency in at least two of molecular biology, protein 
production and crystallographic methods is required for these positions.
The lab is located in the center of Dublin, which is a multi-cultural and 
vibrant city that is well connected to Europe and the USA.
Please email a cover letter, CV, and contact information for three references 
to:
Amir Khan
School of Biochemistry and Immunology
Trinity College Dublin
amir.k...@tcd.iemailto:amir.k...@tcd.ie
http://www.tcd.ie/Biochemistry/research/a_khan/index.php

[ccp4bb] PDF position Structural Cell Biology - Michigan

[Amir Khan]

Hi,
I would like to advertise a position on behalf of Prof. Lois Weisman 
((Michigan).
Interested parties should contact her directly 
(lweisman.off...@gmail.commailto:lweisman.off...@gmail.com).
-Amir

Postdoctoral Fellow Position
Integration of high resolution structures with biology
Seeking a highly motivated postdoctoral fellow to initiate a new project in the 
general areas of phosphoinositide signaling or myosin V-based transport. We are 
a highly multidisciplinary, interactive laboratory, and use diverse techniques 
to address cutting edge questions on the roles and regulation of PtdIns(3,5)P2 
and PtdIns(5)P in yeast and mice. Specifically we are determining how the lipid 
kinase, Fab1/PIKfyve is regulated, identifying the upstream signaling pathways, 
and downstream targets. In addition, we seek to determine the precise roles of 
Fab1/PIKfyve in the nervous system, and how minor perturbations in this pathway 
lead to profound neurodegeneration. Another critical area of research is 
determining how myosin V attaches to cargoes. We recently found that the 
cargo-binding domain of myosin V interacts directly with a subunit of the 
exocyst tethering complex [Jin et al. (2011) Dev. Cell. 21(6):1156-70.].  This 
paper illustrates the power of combining yeast genetics with high-resolution 
structures. Many additional projects in our lab are poised to utilize this type 
of approach.  For an overview of recent projects, see our website:
http://www.lsi.umich.edu/facultyresearch/labs/weisman

Qualifications: A Ph.D. in the life sciences. Experience in molecular, 
biochemical and/or cell biological techniques. A level of publications 
appropriate for the current level of training. The applicant should be 
dedicated to research in the life sciences and have a strong desire to make 
major contributions. Please submit cover letter, CV with name of three 
references, and a brief paragraph indicating a project of interest to 
lweisman.off...@gmail.commailto:lweisman.off...@gmail.com

The University of Michigan is an equal opportunity/affirmative action employer.

[ccp4bb] phaser problem

[Amir Khan]

Hi

Has anyone a solution to the following problem with Phaser (version 2.1.4)?

UNHANDLED ERROR: basic_string::_S_create

It seems to be data and model independent – there was an old thread from 
earlier this year, but I cannot find a follow-up.

Thanks!

Amir

Re: [ccp4bb] Puzzle with MALS

[Amir Khan]

Hi Zhen
-was concentration higher in old run? Might be monomer-dimer equilibrium, 
for recent paper, see Benfield et al. (JBC, in press)
-we have a miniDAWN with a 'low-high-medium' setting in the back of the setting.
Inadvertently changing the setting scales the MW values up and down.
Have you run a BSA standard, to insure system is OK?
-Amir

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of zhen zhang 
[zz2...@columbia.edu]
Sent: Thursday, April 21, 2011 5:32 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Puzzle with MALS

Dear All,

I am having a consistency issue processing my SEC-MALS data. The sample is a 
80KD protein which may form a dimer. The experiment I run a year ago shows a 
major peak at 11.4 ml (GE S200 column), which is calcuated to have a MW of 
144kD. There is a minor peak at 10.0 ml. In an effort to make nice looking 
pictures, I rerun the experiement using the same setup (column, buffer, 
protein, speed) and again the major peak is at 11.4 ml but it is calcuated to 
have a MW of 80KD. I rerun the sample multiple times with the same result. I 
tried to reprocess the old data and the calcuated MW is still 144KD. I am 
puzzled about what I have done wrong and which data I should trust. Well, I am 
inclined to trust the later experiment because I took extra caution and had 
multiple data sets. However, I should be able to reprocess the old data to have 
a MW close to 80KD if I can find out what is wrong and correct the mistake, 
which I have not been able to. Any suggestions?

Zhen Zhang