Re: [ccp4bb] Structural Biology Position at Astex Pharmaceuticals, Cambridge UK

[Anindito Sen]

Dear Puja,

This is Anindito Sen from JEOl Tokyo. I am an  application scientist working 
the field of Cryo-TEM and SEM and a visiting faculty at Bionano department at 
Toyo university Japan. My major research  is focused  on structural 
characterization of protein macro molecules using cryo Electron Microscopy and 
SBF-SEM followed by computational image processing for the last 16 years. I am 
on the final leg of my contract with JEOL and looking for new opportunities. 
There are a few questions regarding the position and was wondering if you can 
help with the following-

Does the candidate needs to be an expert X Ray crystallography or Cryo-EM or 
NMR?

Is this position open for people of any nationality or for people with specific 
 residency/citizenship ? I hold an Indian passport. 

With Best Regards




Anindito Sen. Ph.D
Scientist and Application Specialist in Biological Sciences
JEOL LTD.
13F, Ohtemachi Nomura Bldg.
2-1-1 Ohtemachi, Chiyoda-ku, Tokyo
100-0004
Tel: +81-3-62623563
Fax: +81-3-6262-3577

www.jeol.com



> On Jun 23, 2017, at 7:13 PM, Puja Pathuri <puja.path...@astx.com> wrote:
> 
> Job Title: Structural Biologist (Ref: SB/0617)
> Location: Cambridge, UK
>  
> Astex Pharmaceuticals is a world leader in innovative drug discovery and 
> development. The company has successfully applied its proprietary 
> Fragment-Based Drug Discovery (FBDD) platform to generate multiple new drug 
> candidates that are progressing in clinical development.
>  
> The Molecular Sciences Team is responsible for supporting all activities from 
> ‘gene to structure’ and for prosecuting fragment based screening campaigns. 
>  
> Astex’s Molecular Sciences Team is currently seeking to recruit a talented 
> structural biologist. 
>  
> The role is laboratory based and the successful candidate will be responsible 
> for supporting projects during all phases of the drug discovery process.
>  
> Please find the full advert for the position in the attachment.
>  
> To apply, please send your CV and a cover letter quoting the job reference: 
> SB/0617 to hr...@astx.com <mailto:hr...@astx.com>
>  
> Closing Date – 22nd July, 2017
> * 
> Puja Pathuri, Ph.D.
> Senior Research Associate, Molecular Sciences
> 
> Astex Pharmaceuticals
> 436 Cambridge Science Park
> Milton Road, Cambridge
> CB4 0QA, UK 
> 
> Email: puja.path...@astx.com <mailto:puja.path...@astx.com> 
> Website: www.astx.com <http://www.astx.com/>
> *
>  
> This email and any attachments thereto may contain private, confidential, and 
> privileged material for the sole use of the intended recipient. Any review, 
> copying or distribution of this email (or any attachments thereto) by others 
> is strictly prohibited. If you are not the intended recipient, please delete 
> the original and any copies of this email and any attachments thereto and 
> notify the sender immediately. 

Re: [ccp4bb] Vacancy: structural biologist with cryo-EM expertise at GSK closing date 24th March

[Anindito Sen]

Apologies to you All.

The email got copied to every one. In any case its a junk for rest of you. 
Kindly delete it, if you can.


Best Regards

Andy



Anindito Sen. Ph.D
> On Mar 10, 2017, at 9:17 PM, Vellieux Frédéric <frederic.velli...@ibt.cas.cz> 
> wrote:
> 
> Hello,
> 
> I am not “Dr Chung” and I haven’t received anything from you. Sorry to 
> disappoint you.
>  
> Perhaps a “reply all” isn’t appropriate when replying to a ccp4bb message ? I 
> let you be the judge on that…
>  
> Fred.
>  
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK 
> <mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Anindito Sen
> Sent: Friday, March 10, 2017 12:53 PM
> To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
> Subject: Re: [ccp4bb] Vacancy: structural biologist with cryo-EM expertise at 
> GSK closing date 24th March
>  
> Hello Dr. Chung,
>  
> It was nice talking to you. I have submitted the application online. However 
> I have not received any email conformation. In any case, I am attaching the 
> screenshot of my submission below.
>  
> Kindly acknowledge when you receive my application. 
>  
> Best Regards
>  
> Andy
>  
>  
>  
>  
> 
>  
> Anindito Sen. Ph.D
> Scientist and Application Specialist in Biological Sciences
> JEOL LTD.
> 13F, Ohtemachi Nomura Bldg.
> 2-1-1 Ohtemachi, Chiyoda-ku, Tokyo
> 100-0004
> Tel: +81-3-62623563
> Fax: +81-3-6262-3577
> 
> www.jeol.com <http://www.jeol.com/>
>  
> On Mar 6, 2017, at 7:03 PM, Chun-wa Chung <chun-wa.h.ch...@gsk.com 
> <mailto:chun-wa.h.ch...@gsk.com>> wrote:
>  
> Job-posting  - Seeking structural biologist with cryo-EM expertise at GSK
>  
> A vacancy for a structural biologist with cryo-EM expertise has opened up 
> within the UK structural and biophysical sciences group at GlaxoSmithKline 
> R Joining an experienced multi-disciplinary group, this is an exciting 
> opportunity for a motivated individual to complement and help shape our 
> current capabilities in the EM area. 
> This is a permanent, full-time position suitable for a PhD degree holder (or 
> equivalent).
> 
> To apply for this role and for further details, please click on the link 
> below (Vacancy no : WD106591) 
> https://careers.peopleclick.com/careerscp/client_gsk/external1931/gateway.do?functionName=viewFromLink=330903=en-us
>  
> <https://careers.peopleclick.com/careerscp/client_gsk/external1931/gateway.do?functionName=viewFromLink=330903=en-us>
> A full job description is also included at the end of this message.
> 
> Applications for this vacancy are to be made online by 24th MARCH
> Informal inquiries can be sent to  chun-wa.h.ch...@gsk.com 
> <mailto:chun-wa.h.ch...@gsk.com>
> Thank you for your interest,  
> 
> Chun-wa Chung
> 
> UK Head Structural & Biophysics  Sciences
> GlaxoSmithKline R
> Stevenage
> SG1 2NY
> Tel no: +44 1438 763342
> email : chun-wa.h.ch...@gsk.com <mailto:chun-wa.h.ch...@gsk.com>
>  
>  
>  
> Full Job Description (WD78277):
> GlaxoSmithKline is a world leading research-based pharmaceutical company. We 
> are focused around three core businesses: Pharmaceuticals, Vaccines and GSK 
> Consumer Healthcare. We have a significant global presence with commercial 
> operations in more than 150 countries, a network of 84 manufacturing sites in 
> 36 countries and large R centres in the UK, US,  Belgium and China.
> The UK Structural and Biophysical Sciences group is a multi-disciplinary 
> department within GSK R that provides molecular insights into drug 
> discovery for both small molecule and biopharmaceutical programs.
> 
> We currently have an exciting opportunity for a laboratory-based scientist 
> with expertise in state-of-the-art electron microscopy techniques to play an 
> influential and key role in the application and development of these methods 
> across small molecule and therapeutic protein programs. This individual will 
> have extensive experience in the sample preparation and optimisation 
> processes necessary to produce high-quality molecular images of protein 
> structure, including direct experience of Cryo-EM methodologies. Experience 
> of data analysis and interpretation is also expected. Being responsible for 
> delivering structural insights to support program teams and contributing to 
> the  wider protein structural and biophysical area by identifying and driving 
> forward improvements and innovations that advance drug discovery projects, 
> the individual must be a collaborative team player able to work successfully 
> with other scientists within the department and within diverse teams.
> 
> Key Responsibilities and Accountab

Re: [ccp4bb] Vacancy: structural biologist with cryo-EM expertise at GSK closing date 24th March

[Anindito Sen]

Hello Dr. Chung,

It was nice talking to you. I have submitted the application online. However I 
have not received any email conformation. In any case, I am attaching the 
screenshot of my submission below.

Kindly acknowledge when you receive my application. 

Best Regards

Andy






Anindito Sen. Ph.D
Scientist and Application Specialist in Biological Sciences
JEOL LTD.
13F, Ohtemachi Nomura Bldg.
2-1-1 Ohtemachi, Chiyoda-ku, Tokyo
100-0004
Tel: +81-3-62623563
Fax: +81-3-6262-3577

www.jeol.com

> On Mar 6, 2017, at 7:03 PM, Chun-wa Chung <chun-wa.h.ch...@gsk.com> wrote:
> 
> Job-posting  - Seeking structural biologist with cryo-EM expertise at GSK
>  
> A vacancy for a structural biologist with cryo-EM expertise has opened up 
> within the UK structural and biophysical sciences group at GlaxoSmithKline 
> R Joining an experienced multi-disciplinary group, this is an exciting 
> opportunity for a motivated individual to complement and help shape our 
> current capabilities in the EM area. 
> This is a permanent, full-time position suitable for a PhD degree holder (or 
> equivalent).
> 
> To apply for this role and for further details, please click on the link 
> below (Vacancy no : WD106591) 
> https://careers.peopleclick.com/careerscp/client_gsk/external1931/gateway.do?functionName=viewFromLink=330903=en-us
>  
> <https://careers.peopleclick.com/careerscp/client_gsk/external1931/gateway.do?functionName=viewFromLink=330903=en-us>
> A full job description is also included at the end of this message.
> 
> Applications for this vacancy are to be made online by 24th MARCH
> Informal inquiries can be sent to  chun-wa.h.ch...@gsk.com 
> <mailto:chun-wa.h.ch...@gsk.com>
> Thank you for your interest,  
> 
> Chun-wa Chung
> 
> UK Head Structural & Biophysics  Sciences
> GlaxoSmithKline R
> Stevenage
> SG1 2NY
> Tel no: +44 1438 763342
> email : chun-wa.h.ch...@gsk.com <mailto:chun-wa.h.ch...@gsk.com>
>  
>  
>  
> Full Job Description (WD78277):
> GlaxoSmithKline is a world leading research-based pharmaceutical company. We 
> are focused around three core businesses: Pharmaceuticals, Vaccines and GSK 
> Consumer Healthcare. We have a significant global presence with commercial 
> operations in more than 150 countries, a network of 84 manufacturing sites in 
> 36 countries and large R centres in the UK, US,  Belgium and China.
> The UK Structural and Biophysical Sciences group is a multi-disciplinary 
> department within GSK R that provides molecular insights into drug 
> discovery for both small molecule and biopharmaceutical programs.
> 
> We currently have an exciting opportunity for a laboratory-based scientist 
> with expertise in state-of-the-art electron microscopy techniques to play an 
> influential and key role in the application and development of these methods 
> across small molecule and therapeutic protein programs. This individual will 
> have extensive experience in the sample preparation and optimisation 
> processes necessary to produce high-quality molecular images of protein 
> structure, including direct experience of Cryo-EM methodologies. Experience 
> of data analysis and interpretation is also expected. Being responsible for 
> delivering structural insights to support program teams and contributing to 
> the  wider protein structural and biophysical area by identifying and driving 
> forward improvements and innovations that advance drug discovery projects, 
> the individual must be a collaborative team player able to work successfully 
> with other scientists within the department and within diverse teams.
> 
> Key Responsibilities and Accountabilities: 
> • Design and perform technical experiments in the EM area (sample 
> preparation, optimisation and analysis).
> • Document and communicate scientific results clearly and promptly.
> • Provide strong scientific lead to shape EM strategy within structural 
> biology and across therapeutic discovery programs.
> • Build EM capabilities and infrastructure through interactions with the 
> internal and external scientific community.
> • Maintain scientific excellence and lead in a rapidly evolving area e.g. 
> through interactions such as the FEI/LMB/Pharm consortium.
> • Become an integral team member of multiple project teams.
> 
> Key Capabilities and Proficiency:
> 1. Technical expertise leading to Impact 
> Evidence of specific and in depth knowledge of transmission electron 
> microscopy used as a structural technique, especially single particle 
> cryo-EM. A track record of its application to address scientific problems 
> relevant to pharmaceutical research. 
> 2. Influence and Scientific awareness 
> Evidence of engagement with external scientific environmen

[ccp4bb] Dna model

[Anindito Sen]

Dear All, 

I want to built a 3d model of DNA to be used to show the path of genome 
transfer in a tailed phage electron density map,  during infection. 

It will be helpful if The model can be generated as an mrc  file or of a 
similar file format. 

Any suggestions? 

Thanks 

Andy

Sent from my iPhone

Re: [ccp4bb] [ccp4bb] Remittance advice - Invitation to edit

[Anindito Sen]

my point exactly.

Andy


> On Dec 12, 2016, at 2:49 AM, Laura Spagnolo <laura.spagn...@glasgow.ac.uk> 
> wrote:
> 
> Isn't this mailing list moderated?
> Laura 
> 
> Sent from my iPhone
> 
> On 11 Dec 2016, at 17:48, Bonsor, Daniel <dbon...@som.umaryland.edu 
> <mailto:dbon...@som.umaryland.edu>> wrote:
> 
>> The Nigerian Prince wants your money.
>> 
>> Get Outlook for Android <https://aka.ms/ghei36>
>> From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK 
>> <mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Anindito Sen 
>> <andysen.to...@gmail.com <mailto:andysen.to...@gmail.com>>
>> Sent: Sunday, December 11, 2016 12:45:16 PM
>> To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
>> Subject: [ccp4bb] Fwd: [ccp4bb] Remittance advice - Invitation to edit
>>  
>> Guys 
>> 
>> are you all receiving the same??? What on earth is this ?
>> 
>> Andy
>> 
>> 
>> 
>>> Begin forwarded message:
>>> 
>>> From: Hai-fu Fan <fanha...@gmail.com <mailto:fanha...@gmail.com>>
>>> Subject: [ccp4bb] Remittance advice - Invitation to edit
>>> Date: December 12, 2016 at 2:31:56 AM GMT+9
>>> To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
>>> Reply-To: Hai-fu Fan <fanha...@gmail.com <mailto:fanha...@gmail.com>>
>>> 
>>> Please find attached for your review.
>>> Regards.
>>> 
>>> Hai-fu Fan
>> 
>> 

Re: [ccp4bb] Structural biology software that does not run on Windows or gives important Windows-specific problems

[Anindito Sen]

Just to add to Hernando’s comments
BSOFT and EMAN2 runs on both Macs and LINUX. However its much easier to use 
them on Macs because of the library issues for different flavors of LINUX.

Best 

Andy
 

> On Oct 15, 2016, at 1:31 AM, Hernando J Sosa  
> wrote:
> 
> Most software used for cryo-em analysis (e.g. EMAN2,  Relion, Spider, 
> Frealign etc)  runs better or only in Linux.  
>  
> Best
>  
> Hernando
>  
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK 
> ] On Behalf Of Robbie Joosten
> Sent: Friday, October 14, 2016 11:55 AM
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: Re: [ccp4bb] Structural biology software that does not run on 
> Windows or gives important Windows-specific problems
>  
> Windows 10 now allows a full Ubuntu within Windows. It actually works quite 
> easily.
>  
> There is a good rsync for windows. I use it to make back-ups of the pdb_redo 
> databank. Windows has it own system of incremental backups which works well 
> in my hands. There are two scripting systems in Windows batch and PowerShell. 
> Batchs is fun for simple things, PowerShell is extremely powerful and scarily 
> object oriented for maintenance and administration, not for scientific work.
>  
> That said, I would totally use linux for scientific work, except writing and 
> spreadsheet work.
>  
> Cheers,
> Robbie 
>  
>  
> Sent from my Windows 10 phone
>  
> Van: Tim Gruene 
> Verzonden: vrijdag 14 oktober 2016 17:23
> Aan: CCP4BB@JISCMAIL.AC.UK 
> Onderwerp: Re: [ccp4bb] Structural biology software that does not run on 
> Windows or gives important Windows-specific problems
>  
> Dear Mark,
> 
> XDS does not run on Windows, and even if you take your mtz-file home from the 
> synchrotron nowadays, you sometimes may want to re-refine some data set.
> 
> Windows also costs a lot of money, and if you do a lot of scripting, Windows 
> may be cumbersome. WinCoot is also often mentioned with difficulties that 
> don't occur with Coot. If you like incremental backups to save disk space and 
> time, I am not sure rsnapshot/rsync works with a Windows file system. 
> 
> A windows machine may also impede data exchange with other institutions. Eg. 
> downloading data from https://data.sbgrid.org/  is 
> based on rsync, and if I 
> gave you my usb-stick, Windows would only attempt to format it (well, same 
> would most likely happen with Mac OS X).
> 
> One caveat though: the MS Office version for Mac does not read OpenOffice 
> Format, so I was told.
> 
> Greetings,
> Tim
> 
> On Friday, October 14, 2016 05:14:46 PM Mark J van Raaij wrote:
> > Dear All,
> > 
> > our institution requires me to provide a reasoning not to buy a Windows
> > computer (I want to buy a new MacOSX system), so I am looking for software
> > that does not run or is limited on Windows.
> > 
> > Not available:
> > (Auto)SHARP
> > ARPWARP
> > 
> > Available on Windows but with significant limitations
> > Phenix (no MR-Rosetta, no parallelization)
> > CCP4 (limitations on file-names)
> > 
> > Please correct me if pertinent and provide additional examples if possible.
> > 
> > Gratefully yours,
> > 
> > Mark
> > 
> > Mark J van Raaij
> > Dpto de Estructura de Macromoleculas
> > Centro Nacional de Biotecnologia - CSIC
> > calle Darwin 3
> > E-28049 Madrid, Spain
> > tel. (+34) 91 585 4616
> > http://wwwuser.cnb.csic.es/~mjvanraaij 
> > 
> -- 
> --
> Paul Scherrer Institut
> Dr. Tim Gruene
> - persoenlich -
> Principal Investigator
> Biology and Chemistry
> OFLC/102
> CH-5232 Villigen PSI
> 
> Phone: +41 (0)56 310 5297
> 
> GPG Key ID = A46BEE1A
> 

Re: [ccp4bb] OS X yosemite

[Anindito Sen]

HI Mirek,

You may wanna to check for codes using  libpng12.0.dylib. 

This you have to get from other sources.

Best 

Andy 



Anindito Sen. Ph.D
Scientist  Application Specialist in Biological Sciences
JEOL LTD.
13F, Ohtemachi Nomura Bldg 
2-1-1 Ohtemachi Chiyoda-Ku. Tokyo, Japan
Tel: +81-3-6262-3563
Fax: +81-3-6262-3577

www.jeol.com




 On Mar 18, 2015, at 11:57 AM, William G. Scott wgsc...@ucsc.edu wrote:
 
 Hi Mirek:
 
 I haven’t encountered anything (CCP4, phenix, coot. pymol, etc) that doesn’t 
 work, but also haven’t found a single compelling advantage (or even a 
 non-compelling advantage) to upgrading.
 
 You get to see more of the spinning beachball of death while waiting for file 
 dialog boxes to materialize.
 
 YMMV,
 
 Bill
 
 William G. Scott
 Professor, Department of Chemistry and Biochemistry
 and The Center for the Molecular Biology of RNA
 University of California at Santa Cruz
 Santa Cruz, California 95064  
 USA
 
 http://scottlab.ucsc.edu/~wgscott
 
 On Mar 17, 2015, at 4:30 PM, Cygler, Miroslaw miroslaw.cyg...@usask.ca 
 wrote:
 
 Hi,
 I am thinking of upgrading the os on my mac to Yosemite. Are there any known 
 issues for crystallographic software that I should pay attention to?
 Thanks,
 
 
 Mirek
 
 
 

Re: [ccp4bb] [ot]: nedit on Mac 10.10 yosemite

[Anindito Sen]

Thanks for the note guys. I really love nedit on my Linux boxes but never knew 
that nedit is available for Mac. Can you have the URL for downloading the nedit 
for Mac.

Andy

 Anindito Sen. Ph.D
 Scientist and Application Specialist in Biological Sciences
 JEOL LTD.
 13F, Ohtemachi Nomura Bldg.
 2-1-1 Ohtemachi, Chiyoda-ku, Tokyo
 100-0004
 Tel: +81-3-62623563
 Fax: +81-3-6262-3577
 
 
 www.jeol.com


Sent from my iPad

On 2014/10/30, at 午前12:36, Thomas, Leonard M. lmtho...@ou.edu wrote:

 My guess you might just have to reinstall with the latest version of nedit.  
 That is what I had to do when I went from 10.5 to 10.9.  Have yet to jump to 
 10.10 yet.
 
 
 Leonard M. Thomas Ph.D.
 Macromolecular Crystallography Laboratory Manager
 University of Oklahoma
 Department of Chemistry and Biochemistry
 Stephenson Life Sciences Research Center
 101 Stephenson Parkway
 Norman, OK 73019
 405-325-1126
 lmtho...@ou.edu
 http://barlywine.chem.ou.edu
 http://structuralbiology.ou.edu
 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Ioan Vancea 
 [ivan...@embl-hamburg.de]
 Sent: Wednesday, October 29, 2014 10:00 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] [ot]: nedit on Mac 10.10 yosemite
 
 Hi,
 
 I think that's a standard X11 library, do you have X11 (XQuartz) installed?
 
 Regards,
 Ioan
 
 --
 Dr. Ioan Vancea
 European Molecular Biology Laboratory c/o DESY
 Notkestrasse 85, 22607 Hamburg, Germany
 Tel: +49 (0)40 89902-340
 Email: ivan...@embl-hamburg.de
 
 
 On Oct 29, 2014, at 3:45 PM, Sebastiano Pasqualato wrote:
 
 
 Hi folks,
 sorry for the off-topic and slightly ‘demodée’ question, but, since I 
 updated to Yosemite on my Mac, nedit does not work any more.
 Here’s the error message:
 
 Seba@host041:~ nedit
 dyld: Library not loaded: /usr/X11R6/lib/libXp.6.dylib
  Referenced from: /Applications/nedit/nedit
  Reason: image not found
 Trace/BPT trap: 5
 Seba@host041:~
 
 Anybody knows if there is a fix for that?
 Thanks in advance,
 S
 
 
 
 --
 Sebastiano Pasqualato, PhD
 Crystallography Unit
 Department of Experimental Oncology
 European Institute of Oncology
 IFOM-IEO Campus
 via Adamello, 16
 20139 - Milano
 Italy
 
 tel +39 02 9437 5167
 fax +39 02 9437 5990
 web http://is.gd/IEO_XtalUnit

Re: [ccp4bb] cryo-EM

[Anindito Sen]

Dear Rana,

It appears that you have helical biopolymers formed by the proteins. Given the 
size of 630 kDa as the unit cell or the asymmetric unit forming the biopolymer 
you have a fair chance of gaining insight to the proteins. Using cryo EM  you 
have to collect a sizeable amount of data , followed by computing Fourier 
Transform of the filaments. Using the transform you have to index and determine 
the Axial Rise and turn angle of the filaments. You can then choose any program 
available freely online to calculate the density map of the filaments, thus the 
proteins. You may want to look in 

http://3dem.ucsd.edu/software.shtm

under Helices.

I will recommend  you to use 
BSOFT and FreAlign

www.bsoft.ws
http://grigoriefflab.janelia.org/frealign

Combination of these two yield pretty good results.Keep in mind that such a 
work is a complete project in itself. 

Best wishes





Anindito Sen. Ph.D
Scientist  Application Specialist in Biological Sciences
JEOL LTD.
13F, Ohtemachi Nomura Bldg 
2-1-1 Ohtemachi Chiyoda-Ku. Tokyo, Japan
Tel: +81-3-6262-3563
Fax: +81-3-6262-3577

www.jeol.com




On Aug 30, 2014, at 1:29 AM, rana ibd rna19792...@yahoo.com wrote:

 
 Dear Anindito
 Thank you for your reply and I apologize for writing the wrong name, I work 
 on the HBx proteins which contain molecular weights of (56, 57, 59)kDa 
 including  a tag, but they are all polymers and in size exclusion 
 chromatography for the three proteins give a size of 630kDa
 Best Regards
 Rana
 
 From: Andy Sen andysen.to...@gmail.com
 To: CCP4BB@JISCMAIL.AC.UK 
 Sent: Friday, August 29, 2014 5:06 PM
 Subject: Re: [ccp4bb] cryo-EM
 
 Dear Rana,
 
 Brad's post compelled me to reply to  you. The Cryo EM field is well into its 
 pro stage. 
 
 If you can be specific about the MolWt, size and if it makes any complex, 
 then much precise and helpful answers to your question can be provided. 
 
 Best 
 
 Anindito Sen Ph.D
 JeoL 
 
  On 29/08/2014, at 11:31 pm, Marcus Fislage mf2...@columbia.edu wrote:
  
  Hi Rana,
  
  in contrast to Brads answer, and in case I understand you well, Cryo EM is 
  not in ts infancy stage (as long as you do not talk about Cryo EM on 
  crystals.
  For cryo EM you need only several ul of a nM solution.
  I can always suggest you the 3dem bb. There are of course still 
  limitataions on the size of protein target and resolution, but those are 
  slowly falling.
  
  Some reading material
  http://www.ncbi.nlm.nih.gov/pubmed/23181775
  http://www.ncbi.nlm.nih.gov/pubmed/25071206
  
  
  Cheers
  Marcus
  
  Quoting Brad Bennett bradbennet...@gmail.com:
  
  Yes, but the technique is still in its infancy stage.
  
  DOI: 10.1016/j.sbi.2014.03.004
  
  Cheers-
  Brad
  
  
  On Fri, Aug 29, 2014 at 6:34 AM, rana ibd 
  0285de88044a-dmarc-requ...@jiscmail.ac.uk wrote:
  
  Dear CCP4
  I wanted to ask has anyone tested 3D protein structure by cryo-electron
  microscopy? what is the suitable concentration required for this procedure
  Best Regards
  Rana
  
  
  
  -- 
  Marcus Fislage, PhD
  
  Howard Hughes Medical Institute (HHMI)
  Columbia University
  Department of Biochemistry and Biophysics
  Lab of Joachim Frank
  New York, NY
  
  Phone: 212.305.9524
  Fax: 212.305.9500
 

[ccp4bb] Protein Purification Problem

[Anindito Sen]

Dear All,

This may be slightly off-the-track question but your feedback will be very much 
appreciated. The situation is-

I obtain a very low amount of the protein of my interest (a hetro-dimer) from 
the construct I am using (only 8% of the total amount of protein obtained is 
the protein of my interest).  After 2 column purifications (Ni-NTA and St) the 
concentration of the protein is around 0.24 mg/ml (volume- ~1.0 ml)  from a 
litre of bacterial culture and in ~300 mM NaCl present in the elution  buffer. 

To reduce the high amount of salt I have I use a desalting column which, 
further lowers the protein concentration significantly. 

I need atleast 1.0mg/ml of protein concentration and to the amount of ~200 
microlts for further experiments. 

As the last resort I try to use high amount of bacterial culture (~6lts) to 
scale up the yield and use centricon to concentrate the protein at various 
stages.  I am partially successful to obtain 0.56mg/ml of protein concentration 
and up to  50 microlts of it. 


Another problem is that the protein is  notoriously  prone to  aggregation ( 
1.5 mg/ml), so dialysis for ~ 2 hrs or so to reduce the high salt concentration 
has failed miserably. 

Please do send your feedback.

Thanks and Best Wishes


Andy




Dr. Anindito Sen (Ph.D)
Department of Cell Biology  Anatomy
Graduate School of Medicine
University of Tokyo
Tel  fax: +81-3-5841-3339

Re: [ccp4bb] Protein Purification Problem

[Anindito Sen]

Dear All,

1st of all , Thanks for the very quick feedback. I will answer your questions 
to make the picture more clear-

Nicolas-

Yes the dimer is co-expressed. Almost all the protein is in the sup and not in 
the pellet. Multiple-step  dialysis has failed. I have been trying various 
controls and methods for the last 2 years but of not great success. I do have 
significant amount of protein in the 1st step all including homo-dimers and the 
hetro-dimer. Only 8% of that amount is hetro-dimer. 

Fiona-

I will look in the pI option. Step dialysis has failed due to the aggregation 
issue. St is for Strep affinity. Yes, there is a lot of protein in the flow 
through from the column, I have checked and those protein are the homo-dimers. 

Roger-

I do need to remove/reduce the salt for cryo-Electron Microscopy (cryo-EM). The 
maximum that can be afforded is 50 mM. Glycerol is a big no for cryo-EM so I am 
not sure how to implement it. You are absolutely correct as the membranes of 
the centrifugal concentration devices are creating one big problem. I will try 
PES (polyethylenesulfone) concentrator. The final pH needs to be 6.8-7.0 at the 
for the EM work. So to make a drop from pH 8.0 to 6.8 will be a additive step 
in purification process. Can you suggest how can we do it without much drop in 
the concentration ?

Bryan-

I agree with you that the plasmid I have in my hand is far from being a good 
candidate for the purification process, so I have to venture more. Your point 
No2  3 is well taken, I will look into that. However I cannot have the pH as 
high as 8. It needs to be around 6.8 to 7.0 for EM work. 

Lastly I am not an expert of Protein Purification , my expertise are in Cryo-EM 
and computational  image processing, so I have to go baby-steps of asking such 
questions at the forum. Thanks again. 

Best 

Andy






Dr. Anindito Sen (Ph.D)
Assistant Professor (Project)
Department of Cell Biology  Anatomy
Graduate School of Medicine
University of Tokyo
Tel  fax: +81-3-5841-3339

On Jan 30, 2014, at 10:57 PM, Fiona Whelan fiona.whe...@york.ac.uk wrote:

 Hi Andy,
 
 There are lots of things to try. Here are a few:
 Check your protein heterodimer combined pI, make sure you're at least 1 pH 
 unit away from that for your dialysis step. 
 Perhaps your dimer needs high NaCl to maintain its solubility - try dialysing 
 into a range of NaCl concentrations (100mM, 150, 200, 300 mM). Does it need 
 any divalent cations? Check homologs - do they bind divalent cations - try 
 screening these in your dialysis condition. 
 
 You say 2 column purifications - St - is that Strep affinity? Do you have a 
 lot of protein in the flow through from this column? I've found that using pH 
 8 is very important for binding at this stage - and high protein 
 concentration.
 
 I think the BB might be more helpful with a bit more information about your 
 expression method and if you think the protein is lost at some stage during 
 the purification, or only lost in aggregate after dialysis/during 
 concentration.
 
 Good luck,
 
 Fiona
 
 On 30/01/2014 13:17, Anindito Sen wrote:
 Dear All,
 
 This may be slightly off-the-track question but your feedback will be very 
 much appreciated. The situation is-
 
 I obtain a very low amount of the protein of my interest (a hetro-dimer) 
 from the construct I am using (only 8% of the total amount of protein 
 obtained is the protein of my interest).  After 2 column purifications 
 (Ni-NTA and St) the concentration of the protein is around 0.24 mg/ml 
 (volume- ~1.0 ml)  from a litre of bacterial culture and in ~300 mM NaCl 
 present in the elution  buffer. 
 
 To reduce the high amount of salt I have I use a desalting column which, 
 further lowers the protein concentration significantly. 
 
 I need atleast 1.0mg/ml of protein concentration and to the amount of ~200 
 microlts for further experiments. 
 
 As the last resort I try to use high amount of bacterial culture (~6lts) to 
 scale up the yield and use centricon to concentrate the protein at various 
 stages.  I am partially successful to obtain 0.56mg/ml of protein 
 concentration and up to  50 microlts of it. 
 
 
 Another problem is that the protein is  notoriously  prone to  aggregation 
 ( 1.5 mg/ml), so dialysis for ~ 2 hrs or so to reduce the high salt 
 concentration has failed miserably. 
 
 Please do send your feedback.
 
 Thanks and Best Wishes
 
 
 Andy
 
 
 
 
 Dr. Anindito Sen (Ph.D)
 Department of Cell Biology  Anatomy
 Graduate School of Medicine
 University of Tokyo
 Tel  fax: +81-3-5841-3339
 
 
 
 -- 
 Dr Fiona Whelan
 Potts Group, L1
 Department of Biology
 The University of York
 
 Phone: +44 (0) 1904 328673

Re: [ccp4bb] Docking models into low-res SAD map

[Anindito Sen]

Hi Oliver,

Use CHIMERA- http://www.cgl.ucsf.edu/chimera/

There are video tutorials for docking- 
http://www.cgl.ucsf.edu/chimera/videodoc/videodoc.html

This is a very good package.

While I have used Situs colores, I find this package more versatile and useful. 
You can also use Pymol (but I think thats not free any more??). 

Best 




Dr. Anindito Sen (Ph.D)
Structural Biology
Graduate School of Medicine
University of Tokyo
7-3-1 Hongo. Bunkyo-ku. Tokyo
113-0033. Japan
Tel  Fax: +81-3-5841-3339

On Sep 20, 2013, at 7:09 PM, Randy Read wrote:

 When we have needed to do something like this, the following procedure has 
 worked pretty well for us:
 
 1. Define the volume containing the density into which a model should be 
 docked (most easily as atoms in a PDB file specifying the centres of 
 spheres), then get structure factors corresponding to cut-out density (using 
 cmapcut in CCP4 or phenix.cut_out_density in Phenix).
 
 2. Do a rotation search in Phaser, treating the structure factors from the 
 cut-out density as observed data.
 
 3. Use the phased translation function (program fft in CCP4) to place the 
 oriented model(s) in the cut-out density.
 
 With the availability of cmapcut and phenix.cut_out_density, this is much 
 easier than it used to be, but we've still been intending to streamline this 
 process; this is on our very long to-do list.
 
 In principle, FFFEAR ought to work for any problem where this works, but I 
 haven't had much luck -- probably I've been doing something wrong!
 
 The next time I have a problem like this, I'll have to try Situs colores, 
 which I hadn't heard about until this discussion.
 
 Best wishes,
 
 Randy Read
 
 On 19 Sep 2013, at 16:03, Pete Meyer pame...@mcw.edu wrote:
 
 Another vote for Situs colores - we've had very good luck with using it to 
 dock domain structures into low resolution multi-crystal SAD maps.
 
 Pete
 
 Oliver Clarke wrote:
 Hi all,
 Can anyone recommend software to dock previously solved domains into a 
 (very) low-resolution experimentally phased map?
 I am working on a rather marginal case where this would be useful - very 
 large assembly (500kDA per ASU), native goes to 6.9A, and the best 
 derivative goes to 8A with SAD phases from a heavy atom cluster to 11. 
 Unfortunately no NCS is present.
 The map is (for the resolution!) not too bad - the solvent boundary is 
 clear after SHELXE, and I can manually place a previously solved structure 
 and get a reasonably convincing fit (and the fit I obtain agrees with that 
 previously determined using CryoEM by another group). There are a couple of 
 other domains that I would like to place, and I believe I have some idea of 
 where they are, but manual fitting is somewhat subjective, so I'd like an 
 automated routine for doing the same - something like the functionality 
 that ADP_EM provides when working with CryoEM maps. Does something like 
 this exist? I tried using ADP_EM but it gives a segfault when used with a 
 crystallographic density map.
 I have tried using MOLREP to look for the model in the map to no avail, and 
 PHASER didn't work either.
 On another note, if anyone has any tips for density modification/phase 
 extension in this resolution range I would love to hear them - haven't had 
 a whole lot of luck with PARROT, DM, or SOLOMON, the maps seem stuck at 
 ~10A despite data going to 8. I tried using SHARP with the SPHCLUSTER tag 
 on, but it gave no improvement over what I obtain treating the cluster as a 
 super atom in SHELXD.
 Many thanks in advance!
 Oliver.
 
 --
 Randy J. Read
 Department of Haematology, University of Cambridge
 Cambridge Institute for Medical Research  Tel: + 44 1223 336500
 Wellcome Trust/MRC Building   Fax: + 44 1223 336827
 Hills RoadE-mail: rj...@cam.ac.uk
 Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk

Re: [ccp4bb] Docking models into low-res SAD map

[Anindito Sen]

Hi Folmer,

As I said-  I think its not free any more is indicative that I am not sure of 
its status. In addition to that I do not use  Pymol much. 

Thanks for the correction. 

Best




Dr. Anindito Sen (Ph.D)
Department of Cell Biology  Anatomy
Graduate School of Medicine
University of Tokyo
Tel  fax: +81-3-5841-3339

On Sep 20, 2013, at 8:23 PM, Folmer Fredslund folm...@gmail.com wrote:

 Hi Anindito Sen
 
 
 2013/9/20 Anindito Sen andysen.to...@gmail.com
 skip
  
 . You can also use Pymol (but I think thats not free any more??). 
 
 /skip
 
 
 I do not know what your definition of free is, but PyMOL is an open-source 
 project. You can download the code from http://sourceforge.net/projects/pymol/
 To me, that means free.
 
 It is also possible to pay for PyMOL http://www.pymol.org/pymol which will 
 give you an easy installer for windows and support amongst other things.
 
 Sorry for the OT post,
 Folmer
 
 -- 
 Folmer Fredslund

Re: [ccp4bb] Superposition of electron density

[Anindito Sen]

Hi 

You can use Chimera. 

http://www.cgl.ucsf.edu/chimera/

Best 

Dr. Anindito Sen
Graduate School of Medicine
University of Tokyo
Sent from my iPad

On 2013/08/30, at 午前12:17, Jan Lohöfener lohoefener@mh-hannover.de wrote:

 Dear all,
  
 we are trying to superimpose several structures together with their electron 
 density to show the presence of point mutations. Is there any possibility to 
 superimpose the electron density together with its structure or to 
 translate/rotate the electron density alone? 
  
 Thanks you,
 Jan

[ccp4bb] Insertion of a Tag protein in of the molecules of a Dimer protein complex

[Anindito Sen]

Dear All,

I need to insert a tag protein of ~5 kDa in one of the molecules of a dimer 
protein (size is ~100kDa).  To be more precise -  The tag need to get itself 
attached only on one of the dimer molecules. 

My expertise are not in Cell biology and therefore any suggestion in this 
regard will be of great help.

Thanks  Best Wishes



Dr. Anindito Sen (Ph.D)
Structural Biology
Graduate School of Medicine
University of Tokyo
7-3-1 Hongo. Bunkyo-ku. Tokyo
113-0033. Japan
Tel  Fax: +81-3-5841-3339

[ccp4bb] protein tags

[Anindito Sen]

Dear All,

We are looking to protein tags (of size not exceeding 10 kDa) as markers to 
label our protein complex of size ~50 kDa. Although I have searched for several 
possible candidates mainly mini-proteins, most of them are not what we want to 
have. The features that we are looking in the tag proteins are 


1. Stable   Plus-charged proteins.
   
2. Close distance between N-terminal and C-terminal ends.

3. Possibly with alpha helices.

4. size not exceeding 10 kDa

Any suggestion will be very helpful.

Thanks in advance and Best Wishes 

Andy



Dr. Anindito Sen (Ph.D)
Structural Biology
Graduate School of Medicine
University of Tokyo
7-3-1 Hongo. Bunkyo-ku. Tokyo
113-0033. Japan
Tel  Fax: +81-3-5841-3339