[ccp4bb] Postdoctoral Position in Antibody Design of Ion Channel modulators

[Paul Miller]

Applications are invited for a postdoctoral researcher position to study 
antibody design and molecular mechanisms of modulation of ion channels. You 
will be part of the Modular Activator and Silencer Therapeutics (MAST) team 
developing novel platforms of antibodies, E3 ligase-mediated degraders, and 
proteases, to advance drug discovery, with the opportunity for superb 
mentorship support from the programme, which involves the labs of Laura Itzhaki 
(degraders; PMID 33623657), Paul Miller (ion channel antibodies; PMID 
35140402), Florian Holfelder (proteases; PMID 36765057), Mark Howarth 
(antibodies; PMID 36865256), Catherine Wilson (degraders; PMID 32286286), and 
Pietro Sormanni (antibody design; PMID 37024501)



The applicant will develop synthetic libraries of conformational antibodies 
that can act as modulators of ion channels, identify hits from phage display, 
and demonstrate proof-of-concept modulation by solving cryo-EM structures (and 
by electrophysiology). This project provides opportunities for creative protein 
engineering, including database mining (to build structural antibody 
libraries), machine learning (AI) approaches to antibody design, phage display, 
and cryo-EM of ion channels.



The applicant will have completed a PhD (or soon to be awarded one), and will 
have relevant wet-lab experience that includes protein engineering and/or 
structural biology (cryo-EM or X-ray crystallography) and/or phage display 
and/or computational approaches to analysing protein and antibody structure.



Recent relevant publications are:



Kasaragod VB, Mortensen M, Hardwick SW, Wahid AA, Dorovykh V, Chirgadze DY, 
Smart TG, Miller PS.

Mechanisms of inhibition and activation of extrasynaptic alpha-beta GABAA 
receptors. Nature, 2022, Feb, 602(7897), p. 529-533. PMID: 35140402. 
https://www.nature.com/articles/s41586-022-04402-z



Kasaragod VB, Malinauskas T, Wahid AA, Lengyel J, Knoflach F, Hardwick SW, 
Jones CF, Chen W-N, Lucas X, Omari K, Chirgadze DY, Aricescu AR, Cecere G, 
Hernandez M-C, Miller PS.

The molecular basis of drug selectivity for *5 subunit-containing GABAA 
receptors. Nature Structure and Molecular Biology, 2023, Dec, 30(12), 
p.1936-1946. PMID: 37903907.

https://www.nature.com/articles/s41594-023-01133-1



Ramon, Aubin, Montader Ali, Misha Atkinson, Alessio Saturnino, Kieran Didi, 
Cristina Visentin, Stefano Ricagno, Xing Xu, Matthew Greenig, and Pietro 
Sormanni.

Assessing Antibody and Nanobody Nativeness for Hit Selection and Humanization 
with AbNatiV.

Nature Machine Intelligence, 15 January 2024.

https://doi.org/10.1038/s42256-023-00778-3





Application Closing date: 24th March 2024

Project start date: 14th May 2024 with flexibility in the precise start date 
and continue for 24 months in the first instance.

For more details and to apply: https://www.jobs.cam.ac.uk/job/45358/

Informal enquiries about the position can be made to Dr Paul Miller 
(pm...@cam.ac.uk<mailto:pm...@cam.ac.uk>)

For enquiries about the online application process please contact Debbie 
Stanley, phar...@lists.cam.ac.uk<mailto:phar...@lists.cam.ac.uk>

Paul Miller (he/him), PhD
University Lecturer in Pharmacology
Department of Pharmacology
University of Cambridge
Tennis Court Road
Cambridge
CB2 1PD
+44 1223 761267
https://www.phar.cam.ac.uk/research/miller



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[ccp4bb] Postdoctoral Position in small molecule and antibody modulation of ion channels

[Paul Miller]

Applications are invited for a postdoctoral researcher position in the 
laboratory of Dr Paul Miller at the Department of Pharmacology, University of 
Cambridge, beginning from January – April 2023.


The project will study small molecule and/or antibody modulators of ion 
channels as research tools and therapeutic leads, in order to study channel 
subtype contributions to CNS physiology and disease and empower drug discovery. 
This is a great opportunity to gain experience in one or more aspects of 
handling challenging membrane proteins, understanding small molecule and/or 
antibody modulation, and cryo-EM, ideal for follow-on academic and 
biotechnology careers. The lab utilises mammalian cell protein expression 
systems, membrane protein purification, and cryo-EM via Arctica and Krios 
microscopes with K2/K3 direct detectors, and also has manual and automated 
electrophysiology equipment,. More information about the laboratory is 
available at https://www.phar.cam.ac.uk/research/miller.


The applicant will be about to obtain a PhD or have obtained a PhD within the 
last 5 years. Experience in molecular biology, protein production, purification 
and structural biology (X-ray crystallography or cryo-EM) is required. You must 
be a careful and methodical worker, paying attention to accuracy, be able to 
take responsibility and interact as part of a tightly knit team, and be willing 
to work in a collaborative environment. You should be self-motivated and able 
to organise, perform and analyse experiments with minimal instruction.


A recent relevant publication from the lab is:

Kasaragod VB, Mortensen M, Hardwick SW, Wahid AA, Dorovykh V, Chirgadze DY, 
Smart TG, Miller PS.

Mechanisms of inhibition and activation of extrasynaptic alpha-beta GABA-A 
receptors.

Nature. 2022 Feb;602(7897):529-533. PMID: 35140402

Application Closing date: 30th November 2022
Project start date: January-April 2023
For more details and to apply: https://www.jobs.cam.ac.uk/job/37880/

Informal enquiries about the position can be made to Dr Paul Miller 
(pm...@cam.ac.uk<mailto:pm...@cam.ac.uk>)

For enquiries about the online application process please contact Debbie 
Stanley, phar...@lists.cam.ac.uk


Paul Miller, PhD
University Lecturer in Pharmacology
Department of Pharmacology
University of Cambridge
Tennis Court Road
Cambridge
CB2 1PD
+44 1223 761267
https://www.phar.cam.ac.uk/research/miller



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[ccp4bb] Postdoctoral Position in CNS antibody-ion channel mechanisms and design

[Paul Miller]

Applications are invited for a postdoctoral researcher position in the 
laboratory of Dr Paul Miller at the Department of Pharmacology, University of 
Cambridge, beginning from around October 2022.

The project will focus on studying the molecular basis of antibody modulation 
of ion channel function and applying structure-guided approaches to engineer 
new research tools. This is vital to define channel subtype contributions to 
CNS physiology and disease and to discover new therapeutic modalities. This is 
a great opportunity to gain experience in one or more aspects of handling 
challenging membrane proteins, structure-guided antibody engineering, and 
cryo-EM, ideal for follow-on academic and biotechnology careers. The lab 
utilises mammalian cell protein expression systems, membrane protein 
purification, and cryo-EM via Arctica and Krios microscopes with K2/K3 direct 
detectors, and also has manual and automated electrophysiology equipment,. More 
information about the laboratory is available at 
https://www.phar.cam.ac.uk/research/miller.

The applicant will be about to obtain a PhD or have obtained a PhD within the 
last 3 years. Experience in molecular biology, protein production, purification 
and structural biology (X-ray crystallography or cryo-EM) is required. You must 
be a careful and methodical worker, paying attention to accuracy, be able to 
take responsibility and interact as part of a tightly knit team, and be willing 
to work in a collaborative environment. You should be self-motivated and able 
to organise, perform and analyse experiments with minimal instruction.

A recent relevant publication from the lab is:
Kasaragod VB, Mortensen M, Hardwick SW, Wahid AA, Dorovykh V, Chirgadze DY, 
Smart TG, Miller PS.
Mechanisms of inhibition and activation of extrasynaptic alpha-beta GABA-A 
receptors.
Nature. 2022 Feb;602(7897):529-533. PMID: 35140402

Application Closing date: 15th July 2022
Project start date: Approximately 1st October 2022
For more details and to apply: https://www.https://www.jobs.cam.ac.uk/job/35285/
Informal enquiries about the position can be made to Dr Paul Miller 
(pm...@cam.ac.uk<mailto:pm...@cam.ac.uk>)
For enquiries about the online application process please contact Debbie 
Stanley, phar...@lists.cam.ac.uk


Paul Miller, PhD
University Lecturer in Pharmacology
Department of Pharmacology
University of Cambridge
Tennis Court Road
Cambridge
CB2 1PD
+44 1223 761267
https://www.phar.cam.ac.uk/research/miller



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[ccp4bb] Postdoctoral Position in CNS antibody-receptor mechanisms and design

[Paul Miller]

Dear community

Please see below postdoctoral position available in my lab to perform 
structure-guided engineering of antibody-type modulators of ion channels.

Cheers, Paul

Applications are invited for a BBSRC-funded postdoctoral (research associate) 
position (2-years in the first instance) in the laboratory of Dr Paul Miller at 
the Department of Pharmacology, University of Cambridge, beginning from around 
October 2020.

The project will focus on studying the molecular basis of antibody modulation 
of ion channel function and applying structure-guided approaches to engineer 
unique pharmacological profiles. This research is vital to define the roles of 
specific ion channel subtypes in CNS physiology and disease and to create new 
therapeutic modalities. This is a great opportunity to nurture skills in 
electrophysiology, cryo-EM and structure-guided antibody engineering, ideal for 
follow-on academic and biotechnology careers. The lab utilises both manual and 
automated electrophysiology equipment, diverse mammalian cell protein 
expression systems and purification technologies, and cryo-EM via Arctica and 
Krios microscopes with Falcon 3/K2/K3 direct detectors. More information about 
the laboratory is available at https://www.phar.cam.ac.uk/research/miller.

The applicant must hold a PhD with relevant experience. Experience in ion 
channel structural biology/pharmacology and/or electrophysiology is preferred. 
Experience is sought in one or more aspects of molecular biology, protein 
expression and purification, and structural biology (interpretation of protein 
structure, X-ray crystallography, cryo-EM). You must be a careful and 
methodical worker, paying attention to accuracy, be keen to take responsibility 
as part of a team, and be willing to work in a collaborative environment. You 
should be self-motivated and able to organise, perform and analyse experiments 
with minimal instruction.

Note that the precise wording of the online advert may be a little different.

Application Closing date: 16th July 2021
Project start date: Approximately 1st October 2021
For more details and to apply: https://www.jobs.cam.ac.uk/job/30163/
Informal enquiries about the position can be made to Dr Paul Miller 
(pm...@cam.ac.uk<mailto:pm...@cam.ac.uk>)
For enquiries about the online application process please contact Debbie 
Stanley, phar...@lists.cam.ac.uk




Paul Miller, PhD
University Lecturer in Pharmacology
Department of Pharmacology
University of Cambridge
Tennis Court Road
Cambridge
CB2 1PD
+44 1223 761267
https://www.phar.cam.ac.uk/research/miller



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

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[ccp4bb] Postdoctoral Position in CNS antibody-receptor mechanisms and design

[Paul Miller]

Applications are invited for a postdoctoral (research associate) position 
(18-months in the first instance) in the laboratory of Dr Paul Miller at the 
Department of Pharmacology, University of Cambridge, beginning from 1st 
November 2020.



The project will focus on studying the molecular basis of antibody interactions 
with membrane proteins involved in blood brain barrier transcytosis. This field 
of research is vital for the establishment of therapeutic antibody delivery to 
the central nervous system to treat neurological disorders. This is a great 
opportunity to nurture skills in cryo-EM and/or X-ray crystallography and 
execute structure-guided antibody engineering, ideal for follow-on academic and 
biotechnology careers. The lab utilises automated crystallisation robotics and 
imaging, and new Arctica and Krios microscopes with Falcon 3/K2/K3 direct 
detectors. More information about the laboratory is available at 
https://www.phar.cam.ac.uk/research/miller.



The applicant will hold a PhD with a solid background in structural 
biology/biophysics/biochemistry. Advantageous experience includes: protein 
engineering, production and purification; crystallisation and/or cryo-EM and 
data processing; phage or yeast display platforms; handling membrane proteins. 
You must be a careful and methodical worker, paying attention to accuracy and 
ready to take responsibility as part of a team and willing to work in a 
collaborative environment. You should be self-motivated and able to organise, 
perform and analyse experiments with minimal instruction.



Application Closing date: 9th August 2020

Project start date: 1st November 2020

For more details and to apply: https://www.jobs.cam.ac.uk/job/26225/

Informal enquiries about the position can be made to Dr Paul Miller 
(pm...@cam.ac.uk<mailto:pm...@cam.ac.uk>)

For enquiries about the online application process please contact Debbie 
Stanley, phar...@lists.cam.ac.uk



To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] does 12 A diffraction worth optimization

[Paul Miller]

Absolutely you should optimise! It's impossible to predict crystal behaviour. I 
had a 20 A crystal, and I set a new plate in the same reservoir with an 
additive screen and got a 3A crystal. It was probably a completely different 
crystal to be honest, lattice, etc, but one never knows, you just have to try. 
You can also try using the low res crystal as a seed. Plus a million other 
things.



Paul Steven Miller (PhD)
Postdoctoral Researcher
University of Oxford
Wellcome Trust Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive
Oxford
OX3 7BN


 Original message 
>Date: Fri, 2 Mar 2018 17:34:18 -0800
>From: CCP4 bulletin board  (on behalf of Natalia O 
>)
>Subject: [ccp4bb] does 12 A diffraction worth optimization  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Hello,
>
>    
>
>   I got crystals of protein-nucleic acid complex,
>   rod-shape, reproducible, don’t visibly get damaged
>   upon freezing; however they gave diffraction only to
>   about 12 A. I tried several crystals. My question is
>   whether such crystals worth optimization. Clearly a
>   4A diffracting crystal could potentially be
>   optimized to 3 – 2.5A, but if the diffraction that
>   I am getting now is 12A it could suggest that the
>   system is so flexible that getting to 3A with this
>   crystal form is not possible at all. I just wonder
>   if there is any statistics or a rule of thumb about
>   what initial diffraction worth optimization?
>
>   Thank you!
>
>   -Natalia

Re: [ccp4bb] High R/Rfree after MR

[Paul Miller]

I had a similar problem to what you describe. In my case the dataset was 
severely anisotropic (2.6A, 3.4A, 4.6A in each axis). The R factors were stuck 
similar to yours but the map looked good. I was told by someone with a much 
better appreciation of the theory than myself that the anisotropy was causing 
the problem.

It would be interesting to know from an expert in anisotropy e.g. the creators 
of UCLA anisotropy server or Startaniso whether anisotropy can cause this 
problem and whether there is any way around it.

Cheers, Paul

Paul Steven Miller (PhD)
Postdoctoral Researcher
University of Oxford
Wellcome Trust Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive
Oxford
OX3 7BN


 Original message 
>Date: Fri, 13 Oct 2017 10:30:22 +0200
>From: CCP4 bulletin board  (on behalf of Gianluca Cioci 
>)
>Subject: [ccp4bb] High R/Rfree after MR  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Dear All,
>   I am trying to refine a structure at 3.3A. Model has
>   60% identity to the target. Maps look OK (for 3.3A)
>   and rebuilding in Coot is relatively
>   straightforward. However, after some rebuilding
>   cycles the R factors are stuck at 0.37/0.39
>   (REFMAC). 
>   XTRIAGE tells me that everything is normal (no twin,
>   98% completeness, R=3.5% in the low resolution bin),
>   perhaps some anisotropy is present. 
>   I have already refined 2 homologous structures at
>   resolutions going from 3.2 to 3.8 and there were no
>   problems (final R ~ 0.21/0.24). 
>   Any advice ?
>   Thanks,
>   GIA

Re: [ccp4bb] protein precipitation reg

[Paul Miller]

Only one of your dialysis buffers has a decent (100 mM) NaCl. Maybe you could 
try higher salt IN COMBINATION with glycerol. There's also NDSBs that stabilise 
proteins.

Also, could the high immidazole be keeping the protein happy? You could test 
this by dialysing into the same buffer with a range of immidazole 
concentrations. You could try other related natural compounds, histidine, 
histamine, as well to understand this process better.

Cheers, Paul

Paul Steven Miller (PhD)
Postdoctoral Researcher
University of Oxford
Wellcome Trust Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive
Oxford
OX3 7BN


 Original message 
>Date: Thu, 30 Mar 2017 11:32:05 +0530
>From: CCP4 bulletin board  (on behalf of Akilandeswari 
>Gopalan )
>Subject: Re: [ccp4bb] protein precipitation reg  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Dear all,
>   I have used the following buffers for purification
>   and dialysis. this is fyi.
>
>   Lysis buffer:
>
>   25mM Tris pH 7 or 7.5 or 8
>
>   100-500mM NaCl (increase in salt concentration
>   increased precipitation of the protein in the column
>   itself)
>
>    5mM Beta mercaptoethanol 
>
>   0.5% Triton X 100 
>
>   I have tried with other buffers also.
>
>   a. HEPES buffer pH7.5
>
>   b. Phosphate buffer pH 7.8
>
>   c. MOPS buffer pH 8
>
>    
>
>   Wash and Elution Buffer:
>
>   25mM Tris pH 7 or 7.5 or 8
>
>   100-500mM NaCl 
>
>   20 and 30mM Imidazole for wash
>
>   300mM for elution
>
>    
>
>    
>
>   Dialysis Buffer:
>
>   1. Tris 25mM pH 7
>
>   2. Tris 25mM pH 7.5
>
>   3. Tris 25mM pH 8
>
>   4. Tris 25mM pH 7.5, 5% glycerol
>
>   5. Tris 25mM pH 7.5, 10% glycerol
>
>   6. Tris 25mM pH 7.5, 20% glycerol
>
>   7. Tris 25mM pH7.5, 50mM NaCl
>
>   8. Tris 25mM pH7.5, 100mM NaCl
>
>   9. Tris 25mM pH7.5, 1mM MgCl[2
>
>   ]10.  Tris 25mM pH7.5, 50mM NaCl, 50mM L-Arg, 50mM
>   L-Glu
>
>   In all these cases the protein precipitates. i have
>   tried to do buffer exchange also. i can see
>   precipitate sticking on the walls of the tube during
>   the process. 
>
>   On Thu, Mar 30, 2017 at 10:14 AM, Debanu Das
>    wrote:
>
> Hi Akila,
>
> In addition to what others have asked about the
> dialysis buffer, a few
> more comments that might help to decide next steps
> because the
> precipitation (note precipitation and aggregation
> are related but not
> synonymous) may be due to several different or
> related reasons:
>
> 1) At what stage are you dialyzing? Is it after
> SEC? Could your
> protein be too concentrated at that point since
> your yield is high
> leading to some precipitation? How severe is the
> loss?
> 2) Did you try more purification before dialysis?
> 3) Are you removing the detergent in the buffer
> you are dialyzing against?
> 4) Can you try buffer exchange during
> concentration instead of dialysis?
> 5) Try increasing your NaCl concentration and
> adding 5-10% glycerol to
> improve protein solubility.
> 6) Did you try cleaving the C-term His-tag before
> dialysis? Did you
> try N-term His tag?
> 7) Do you really need to dialyze? Did you try
> assays or
> crystallization trials with the purification
> buffer? You can run a SEC
> column without imidazole to remove that before
> crystallization.
> 8) PSI/SBKB TargetTrack can be great resource to
> look at
> expression/purification protocols for similar
> proteins:
> http://sbkb.org/tt/
> 9) Also look up the Tb Structural Genomics
> resource to see if there is
> anything on this target or related targets:
> http://www.webtb.org/
>
> Best,
> Debanu
>
> On Wed, Mar 29, 2017 at 6:38 AM, Akilandeswari
> Gopalan
>  wrote:
> > Dear all,
> > I am a PhD student doing structural studies on a
> few proteins from
> > Mycobacterium tuberculosis. The gene encoding
> the proteins I work on are
> > cloned into pet22b with c terminal His tag. the
> proteins are expressing
> > well. upon purification I am getting good yield
> of protein but during
> > dialysis, the proteins precipitate. Kindly
> suggest some solutions to avoid
> > aggregation. pI of one protein is 9.7 and that
> of the other is 5.6
> > I am using 25mM Tris pH 7.5 and 100 mM NaCl
> buffer with 5mM
> > beta-mercaptoethanol and 0.5% triton x 100 for
> lysis, the same buffer with
> > 20-30mM imidazole for washing and 300mM
> imidazole for eluting the proteins.
> >
> > Thank you
> > Regards
> > Akila
> >
> > --
> > Akilandeswari G
> >
>
>   --
>   Akilandeswari G
>   JRF
>   C/O Dr. Alamelu Raja
>   National Institute for Research in Tuberculosis
>   Chetput, Chennai