[ccp4bb] PhD (CIFRE) position – SANOFI & ESRF on cryoEM methods applied to antibodies

[Magali Mathieu]

PhD (CIFRE) position – SANOFI & ESRF on cryoEM methods applied to antibodies



PhD thesis title: High resolution Cryo- Electron Microscopy of Antibody / 
Antigen complexes
More and more research focus is being put on biologics and their development as 
drugs. These molecules are usually large and flexible and they can interact 
with various targets located on the surface of cells; Bi-specific antibodies 
are an example that can potentially bind to two different antigens and which 
have gained attention due to their potential application in cancer 
immunotherapy and drug delivery. To fully understand their mechanism, 
structural knowledge at an atomic level is needed.

While low resolution studies can be obtained using traditional microscopy, high 
resolution studies of these interactions are usually performed by protein 
crystallography (or by Hydrogen Deuterium Exchange studies) using fragments of 
both antibody and antigen. The ESRF is in progress of installing a Krios 
microscope which, coupled with a direct electron detector and a Volta phase 
plate will enable us to determine the full structure of the complexes.
The successful candidate will characterise Ag/Biologics complexes from Sanofi, 
using state-of-the-art microscopes at the cryo-EM platform of the Partnership 
for Structural Biology (PSB) and will determine the high-resolution structure 
of one or more of these complexes.
The PhD thesis will last 3 years starting in late autumn 2017, in a 
collaboration between Sanofi (Vitry sur Seine, France) and the ESRF (Grenoble, 
France) under the co-supervision of Dr. Magali Mathieu (Sanofi) and Dr. Gordon 
Leonard (ESRF). This thesis will be CIFRE (Conventions Industrielles de 
Formation par la REcherche) and the student will be a Sanofi employee (CDD) for 
the whole duration of the thesis. Most of his/her work will be performed at the 
ESRF.

The applicant must have a Master Degree (Master2 or MSc) in integrated 
structural biology or biophysics, some knowledge in cryo-electron microscopy 
and in molecular and cell biology. He/she should be a team player and have good 
communication skills.

CV and accompanying letter should be sent to 
magali.math...@sanofi.com AND 
christoph.mueller_dieckm...@esrf.fr 
before July 7, 2017.




Magali Mathieu
Sanofi
Bio Structure and Biophysics / iDD France
Centre de Recherche de Vitry/Alfortville
TÉL. : +33 (0) 1.58.93.39.90  -  FAX : +33 (0) 1.58.93.80.63
13, quai Jules Guesde – BP 14 – 94403 Vitry-sur-Seine Cedex France

[cid:image001.jpg@01D28B97.134FDFD0]

Re: [ccp4bb] turn off probe clashes in stand alone Coot

[Paul Emsley]

On 01/06/2017 23:39, Ursula Schulze-Gahmen wrote:
Does anybody know how to turn off the molprobity probe clashes in the stand-alone version of 
Coot? I just turned them on and now I seem to be stuck with them.


Draw -> Generic Display Objects -> Undisplay All

or Delete the if you wish.

Paul.

[ccp4bb] turn off probe clashes in stand alone Coot

[Ursula Schulze-Gahmen]

Does anybody know how to turn off the molprobity probe clashes in the
stand-alone version of Coot? I just turned them on and now I seem to be
stuck with them.

Ursula

-- 
Ursula Schulze-Gahmen, Ph.D.
Project Scientist
UC Berkeley, QB3
360 Stanley Hall #3220
Berkeley, CA 94720-3220
(510) 643 9491

[ccp4bb] Postdoc position at King's College London

[Anna Davies]

Dear All,

A short-term (maternity cover) postdoctoral position is available in 
protein X-ray crystallography on a drug discovery project at King's 
College London (Guy's campus, London, UK).


The following link contains further information about the post, and 
details on how to apply:


http://www.jobs.ac.uk/job/BBT944/research-associate/

Informal enquiries can be made to Prof. Brian Sutton 
(brian.sut...@kcl.ac.uk).


The closing date for applications is 14 June 2017.

Thanks,

Anna

-
Dr Anna Marie Davies
Randall Division of Cell and Molecular Biophysics
King's College London
New Hunt's House
Guy's Campus
London
SE1 1UL
United Kingdom

Tel: +44(0)207-848-6419
Email: anna.dav...@kcl.ac.uk

[ccp4bb] Post-doc in membrane protein structural biology, Karolinska Institute.

[Joseph Brock]

Dear colleagues,

Please see the information below regarding an opening in our research group. 
Please share with anyone who might be interested.

Best regards,

-Joseph.



A two-year post-doctoral position is available at the Department of Medical 
Biochemistry and Biophysics (MBB), Karolinska Institutet, Stockholm. The 
project deals primarily with over-expression, purification, crystallisation and 
structure determination of human membrane proteins involved in the generation 
of lipid mediators of inflammation. The successful candidate will join a 
multi-disciplinary research team and the position represents an opportunity for 
a postdoctoral scientist to develop their expertise as well as broaden their 
experience. The major interests of the lab are in membrane protein structure 
and function. We combine structural techniques such as macromolecular 
crystallography with biophysical, biochemical and computational methods to 
investigate the structural basis of catalysis. The lab has all necessary 
equipment and access to facilities needed for the project, including a core 
facility for protein science. We have periodic access to beamlines at ESRF 
(France), Diamond (UK), BESSY and DESY (Germany) and also have access to 
state-of-art cryo-EM facilities.

Qualifications:
The candidate should hold a PhD conferred within the previous two years (or 
expect conferral in the near future), be motivated to independently solve 
problems and a have a strong background in biochemistry or structural biology. 
Experience with protein expression, purification and biochemical 
characterisation is required.  Prior experience in membrane protein 
biochemistry and computational skills such as a familiarity with the LINUX 
operating system will be added advantages.

The application should contain a single PDF document including:
·   Letter of intention stating the scientific interests, technical skills, 
and previous experience of the candidate.
·   CV including date and field of (expected) graduation and list of 
publications, with the contact information of at least two references.

This should be sent to joseph.br...@ki.se and jesper.haeggst...@ki.se.

The deadline for application is the 22nd of June, 2017.



Joseph Brock | PhD
Division of Physiological Chemistry II
Department of Medical Biochemistry and Biophysics
Karolinska Institutet
Scheeles väg 2
SE-171 77 Stockholm, Sweden

Re: [ccp4bb] help needed to interpret a SRF

[R. Michael Garavito]

Vincent,I see a few of problems with your SRF (the maps) which would impact the interpretation.  First you say that both crystals are processed as P21, which you would expect a very strong peak (100% of your origin peak) on kappa/chi=180 at the b* axis (one of the major axes of your map); this arises from the crystallographic symmetry.  Where this axis is depends on what the conventions are of the program you are using. Your SRF has a major peak along Z, but is that the b* axis?  (My b* axis is always placed North-South or your X, á la the Rossmann conventions.  I don’t know the conventions for Molrep) Then you see major off-axial peaks.  The sad thing about your off-axial peaks is that they are badly split.The second problem is that the second crystal shows very strong peak (1001% of your origin peak) on kappa/chi=180, but at different position (along Y).  The control peak is the expected peak which arises from the P21 crystallographic symmetry.  If the data sets were processed as P21, they should be indexed with the 2-fold axis along b/b*, thus the control peak is the expected peak which arises from the P21 crystallographic symmetry.  Once that peak appears in the same place on each map, you can then compare the maps with more confidence.The third problem is that all your maps have the “appearance" of mirror symmetry across the x-axis, which would be expected as it is the consequence of the P2(1) symmetry and you are looking at a hemisphere.  But it suggests that the 2-fold axis is along Y (b/b*). Then map 1 is missing the expected peak on kappa/chi=180 at the b* axis.Final comment is that the maps labeling suggest that the radius of integration is 62-66 Å, which is way, way too large, even for an empty unit cell.  If it is, reduce it down to 20-30 Å to avoid intermolecular cross-peaks and see if the maps become clearer.  I have attached a SRF map from a P21 crystal form (radius of integration = 20 Å, resol. = 2.8 Å) with the 2-fold axis indexed along b/b* (Y) from polarrfn; note the “appearance" of mirror symmetry perpendicular to across the b/b*-axis (North-South or y-axis).Cheers,Michael

EhaAc_kappa180.pdf
Description: Adobe PDF document

R. Michael Garavito, Ph.D.Professor of Biochemistry & Molecular Biology603 Wilson Rd., Rm. 513   Michigan State University      East Lansing, MI 48824-1319Office:  (517) 355-9724     Lab:  (517) 353-9125FAX:  (517) 353-9334        Email:  rmgarav...@gmail.com

On Jun 1, 2017, at 4:51 AM, vincent Chaptal  wrote:Dear Manfred, attached are the postcript files. VincentOn 01/06/2017 10:19, Manfred S. Weiss wrote:Dear Vincent,it would be good if you post the postscript file as well.It is called molrep_rf.ps or something like that. Cheers, ManfredAm 01.06.2017 um 10:12 schrieb vincent Chaptal:Thank you for your email. the anisotropic resolutions of the datasets are 5.6-7.1A for the best and worst diffracting directions of the crystal without additive, and 4.0-5.8A for the crystal with additive. The two crystals come from the same prep and same drop setup, only differ from the presence of the additive during crystallogenesis. They are indeed two different crystals, I would be curious to know more how to compare these two datasets together as I thought it was not possible with such different cell parameters.  On 31/05/2017 17:12, Eleanor Dodson wrote:Well - you dont give details o f resolution but the sovent content and the peak of 0.51 would suggest a possible dimer in crystal 1Crystal 2 is so different it might well have a dimer in a different orientation.Yes, it could very well be the case. But wouldn't there be a peak as well in the SRF? I would try to see if there was a relationship between Xtal 1 and Xtal 2 - can tell you how I would do that if you are interested..But doesnt mass spec or some such technique suggest whether there is a dimer or not?I am not aware of a way to test by Mass Spec or other techniques the content of the ASU, I would be very interested if anyone can further my knowledge on this. All the bestVincentEleanorSelf Rotation Functions are a) hard to interpret and b) often misleading! On 31 May 2017 at 14:18, vincent Chaptal  wrote:Dear all, I need help interpreting results from a SRF; I am very naïve at interpreting them and would appreciate any pointer... I have 2 crystals, before and after additive during crystallogenesis. They have different cell parameters, and I am wondering if I have a monomer or a dimer in the ASU, and if the additive changed this. crystal w/o additive: P21  114,5  107,6  134,6 beta=95,74    1monomer in the ASU = 80% solvent, 1 dimer in the ASU = 61% solvent. Note that this high solvent content agrees well with the fact that this is a membrane protein, and diffracts both to low resolution and very anisotropic...     SRF from Molrep:+

Re: [ccp4bb] help needed to interpret a SRF

[vincent Chaptal]

Thank you for your email.

the anisotropic resolutions of the datasets are 5.6-7.1A for the best 
and worst diffracting directions of the crystal without additive, and 
4.0-5.8A for the crystal with additive.


The two crystals come from the same prep and same drop setup, only 
differ from the presence of the additive during crystallogenesis. They 
are indeed two different crystals, I would be curious to know more how 
to compare these two datasets together as I thought it was not possible 
with such different cell parameters.


On 31/05/2017 17:12, Eleanor Dodson wrote:
Well - you dont give details o f resolution but the sovent content and 
the peak of 0.51 would suggest a possible dimer in crystal 1



Crystal 2 is so different it might well have a dimer in a different 
orientation.
Yes, it could very well be the case. But wouldn't there be a peak as 
well in the SRF?


I would try to see if there was a relationship between Xtal 1 and Xtal 
2 - can tell you how I would do that if you are interested..
But doesnt mass spec or some such technique suggest whether there is a 
dimer or not?
I am not aware of a way to test by Mass Spec or other techniques the 
content of the ASU, I would be very interested if anyone can further my 
knowledge on this.


All the best
Vincent


Eleanor
Self Rotation Functions are a) hard to interpret and b) often misleading!


On 31 May 2017 at 14:18, vincent Chaptal > wrote:


Dear all,

I need help interpreting results from a SRF; I am very naïve at
interpreting them and would appreciate any pointer...

I have 2 crystals, before and after additive during
crystallogenesis. They have different cell parameters, and I am
wondering if I have a monomer or a dimer in the ASU, and if the
additive changed this.

*crystal w/o additive:*
P21  114,5  107,6  134,6 beta=95,74
1monomer in the ASU = 80% solvent, 1 dimer in the ASU = 61%
solvent. Note that this high solvent content agrees well with the
fact that this is a membrane protein, and diffracts both to low
resolution and very anisotropic...
SRF from Molrep:

+--+

| theta phi chi P(i)/P(0)|

+--+

| 1 0.00 0.00 0.00 1.00 |

| 2 148.48 0.00 180.00 0.51 |

| 3 161.29 0.00 180.00 0.31 |

| 4 105.34 180.00 180.00 0.30 |

| 5 74.78 -56.26 179.58 0.27 |

| 6 15.14 -163.78 180.00 0.25 |

| 7 67.61 -40.16 180.00 0.24 |

| 8 134.54 -180.00 180.00 0.19 |

| 9 72.33 -37.82 180.00 0.18 |

| 10 69.72 38.92 175.62 0.18 |

| 11 110.28 -141.08 175.62 0.18 |

| 12 96.58 101.90 179.79 0.18 |

| 13 67.04 -15.02 179.71 0.18 |

| 14 62.76 -15.52 179.45 0.17 |

| 15 117.26 164.48 179.45 0.17 |

| 16 68.39 -18.92 179.86 0.17 |

| 17 70.52 -16.51 180.00 0.17 |

| 18 24.05 -162.02 179.98 0.17 |

| 19 78.61 -36.36 180.00 0.17 |

| 20 83.17 78.42 173.34 0.16 |

| 21 96.83 -101.58 173.34 0.16 |

| 22 81.25 -75.03 179.73 0.16 |

| 23 81.81 -17.73 180.00 0.16 |

| 24 11.02 -142.22 180.00 0.16 |

| 25 76.14 -16.64 179.71 0.16 |

| 26 76.56 -16.93 180.00 0.16 |

| 27 162.29 32.44 180.00 0.16 |

| 28 10.03 -136.82 180.00 0.15 |

| 29 68.67 -58.10 179.49 0.15 |

| 30 111.33 121.90 179.49 0.15 |

| 31 62.99 -20.19 180.00 0.14 |

| 32 97.24 145.00 179.78 0.14 |

| 33 99.56 165.86 179.74 0.14 |

| 34 84.27 -82.68 180.00 0.14 |

| 35 84.51 72.76 174.42 0.13 |

+--+



Crystal with additive:
P21

CELL 117.2840 110.3330 155.6880 90. 93.4280 90.

1 monomer in the ASU = 84% solvent, 1 dimer = 68% solvent.

SRF from Molrep:

+--+

| theta phi chi P(i)/P(0)|

+--+

| 1 0.00 0.00 0.00 1.00 |

| 2 59.55 0.00 180.00 0.36 |

| 3 156.11 0.00 180.00 0.29 |

| 4 10.00 -165.58 180.00 0.26 |

| 5 110.22 -180.00 180.00 0.26 |

| 6 12.97 -169.15 180.00 0.23 |

| 7 166.21 -9.06 180.00 0.22 |

| 8 81.64 -9.61 179.74 0.21 |

| 9 86.02 80.72 178.22 0.20 |

| 10 93.98 -99.28 178.22 0.20 |

| 11 5.96 -54.00 180.00 0.19 |

| 12 67.40 -10.81 180.00 0.19 |

| 13 87.10 -94.56 179.91 0.19 |

| 14 147.13 47.70 10.71 0.19 |

| 15 147.13 -47.70 10.71 0.19 |

| 16 87.45 91.04 155.74 0.19 |

| 17 92.55 -88.96 155.74 0.19 |

| 18 5.33 -63.00 180.00 0.19 |

| 19 104.60 169.61 179.59 0.19 |

| 20 88.01 -85.32 179.05 0.18 |

| 21 92.02 94.64 179.68 0.18 |

| 22 87.61 91.31 153.16 0.18 |

| 23 92.39 -88.69 153.16 0.18 |

| 24 6.65 -46.80 180.00 0.18 |

| 25 153.26 36.01 21.16 0.18 |

| 26 153.26 -36.01 21.16 0.18 |

| 27 63.11 -11.30 179.51 0.18 |

| 28 76.82 -5.09 179.61 0.18 |

| 29 87.69 91.37 148.72 0.18 |

| 30 9