[ccp4bb] PhD project opportunity

[Rafael Guimaraes da Silva]

PhD project available through EASTBIO
Reconstruction, enzymology and structural biology of extinct ancestral enzymes
https://synergy.st-andrews.ac.uk/dasilva/join-us/


___
Dr Rafael G da Silva
Biomedical Sciences Research Complex
School of Biology
Faculty of Science
University of St Andrews
North Haugh, BMS Annexe B103
St Andrews, Fife KY16 9ST UK
Tel: +44 01334 463496
http://synergy.st-andrews.ac.uk/dasilva/




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Re: [ccp4bb] R-merge is too high !!

[Zhang Foggy]

Dear All,

Sorry for the late reply. I was out of town in last two weeks.

Again, thanks for your kindly comments, and here are the summary for the
additional comments and my responses:

1. Summary:

The high value of the R-merge might be due to the wrong orientation matrix
(beam center), anisotropic, potential sample vibration, or radiation damage.

The suggestions are checking the orientation matrix and beam position, and
use pointless "centre" option in XDS to correct the center, use only
partially frames to do integration,  or  avoid sample vibration.

2. Responses to the comments:

(1). Dr. Klaus Futterer suspected the data has large variation during the
lattice parameters refinement or orientation matrix coefficients during
integration, and suggested to determine the orientation matrix in XDS with
all frames or only part (10-20%) of the frames with clear diffraction
spots, and then integrate the frames without floating the lattice
parameters.

Response: Thanks for the suggestion, I will work on this soon and see what
is going to happen.

(2). Dr. Eric Montemayor suspected the data is anisotropic, and suggested
to submit the data to the staraniso server.

Response: I submit the data to the anisotropic server (
http://services.mbi.ucla.edu/anisoscale/), and you can see in the
attachment that my data only has low and mild anisotropy.

[image: anisotropy.png]

(3). Dr. James Holton suspected that current results could be caused by
sample vibration due to particular crystal and loop shape (reference: J.
Appl. Cryst., 2008, 41, 1122). (I) Ways to diagnose sample vibration: (a)
Look at the highest intensity bin to see Rmerge for the brightest spots;
(b) Take a series of "stills" or at least the same narrow rotation about
3-10 times in a row from the same crystal with the same starting phi each
time, integrate the images as usual, and look at the variation (Rmerge) for
each hkl individually. Then plot the variation vs. location on the
detector. If we see swaths of reciprocal space with high variation while
others low, and the orientation of the swaths is not related to the phi
axis, then we know we've got vibration issues. (c) Look at other data sets
from the same instrument. If the low-angle Rmerge in P1 of the dataset
stands out, then it was probably sample vibration. (II) Ways to solve this
issue (a) adjust the flow rate of the cold stream; (b) shore-up the mounts;
(c) coat the twisty loop neck with epoxy before mounting the crystal; (d)
use loop and base (curved) from MiTeGen (MiTeGen must pay the advertising
fee!). (III) If Rmerge is high like I am seeing here I should definitely
inform whomever is maintaining the instrument I used that there is a
problem.

Response: Thanks for the suggestion. Actually, I have discussed with the
beamline stuff to argue this potential issue. However, (a) after collecting
this set of data, I do collect data from other crystals grown from
different protein, and the R-merge looks fine. (b) I have collected four
sets of data again four months later, and all of them show similar high
R-merge value.  The stuff thought the high R-merge value of the crystals
from only this type of protein should not due to the hardware of the
synchrotron nor the loop itself.

(4). Dr. Thomas Cleveland mentioned that he had a similar situation before,
and this could be caused by a misindexing of the diffraction origin by +/-
1 due to a slightly incorrect beam center. He suggested to use the
pointless "centre" option in XDS to do a search for the correct center just
like the example at
https://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Pointless, and
then use the INDEX_ORIGIN parameter to specify the correct origin, and
reintegrate the data.

Response: Thanks for the suggestion, I will work on this soon and see what
is going to happen.

(5). Dr. Kajander, Tommi A asked that if I can see R-merge difference if I
only process a sector from different place or lower the given symmetry for
the redundancy issue, and if I have tried XSCALE after XDS and crystal name
for correction to look at low resolution binds. He suggested that I need to
check the direct beam position as well as the radiation damage.

Response: I do the integration under P1 or only scale part of the frame,
but the R-merge increased to over 45% very soon.  Regarding to the
radiation damage issue, I do see high radiation damage. However, due to the
weak diffraction, I have to increase the exposure time to get the
resolution. Anyway, I will try to use XDS to correct the beam position and
use partial frames to redo the integration.


Thank you for your kindly help again, and I will update once I process the
data by using XDS to correct the beam position later!

Best,

Liang

Kajander, Tommi A  于2018年10月7日周日 上午3:42写道：

> on the redundancy issue - if you lower it given high symmetry or process
> only a sector from different place - no difference?
>
> i think one suggestion was misindexing by 1 in some direction - did you
> check 

[ccp4bb]

[Renato Mateus Domingos]

Hi all

I am trying to Mutate a Cys-SH to Cys-SO3.

I was replacing CYS with a OCS, using Coot:

1) centre on the CYS of interest
2 ) Extensions -> Modelling -> Replace Residue ... -> OCS -> Mutate

However the residue appears disconnected from the main chain, and I could
not find a way to make the proper bonds.

Anyone can help?

Renato
*Renato Mateus Domingos*
*PhD student at Departamento de Genética e Biologia Evolutiva*
*Universidade de São Paulo, Brazil*
*Phone: (+55)(11)30917590*



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[ccp4bb] Open postdoc position, University of Pennsylvania School of Medicine

[Yi-Wei Chang]

Dear all,

The Yi-Wei Chang lab in the University of Pennsylvania School of Medicine is 
looking for an enthusiastic postdoc to join the group. Our main research 
interests are (1) in vivo structural study of important biological 
processes/nanomachines through electron cryo-tomography; and (2) development of 
correlative light and electron microscopy methods and further their 
applications. (Our lab website)

The successful candidate is expected to have a strong background in cryo-EM, 
X-ray crystallography, super-resolution microscopy, physics, or computer 
programming, and, most importantly, is inspired by the importance and FUN of in 
situ structural determination.

Our institute is fully equipped for cutting-edge electron cryo-tomography: a 
300keV Titan Krios G3i cryo-EM with Volta phase plate, Quantum energy filter 
and K3 direct detector, a cryo-FIB-SEM, a cryogenic fluorescence light 
microscope, a comprehensive sample preparation and screening facility 
(EMRL), and several 
state-of-the-art computer clusters for data processing.

If you are interested in joining us and having a lot of fun together while 
building up a strong publication record, please send your CV and a cover letter 
to me directly at 
(yi-wei.ch...@pennmedicine.upenn.edu).
 Please also feel free to contact with me if you have any questions regarding 
this position. Thanks!

Sincerely,
Yi-Wei

—
Yi-Wei Chang, Ph.D.
Assistant Professor
Dept. of Biochemistry & Biophysics
Perelman School of Medicine
University of Pennsylvania
913B Stellar-Chance Labs
422 Curie Blvd.
Philadelphia, PA 19104-6059
(Office) 215-898-7789
(Lab) 215-898-1191
www.yiweichanglab.org




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[ccp4bb] Research Institute Manager post

[Ragan, Tj (Dr.)]

The Leicester Institute of Structural and Chemical Biology (LISCB) is one of 
five flagship research institutes recently established by the University of 
Leicester. LISCB hosts 22 active research groups and brings together 
internationally renowned research using structural biology, chemical biology 
and single molecule methods to investigate major challenges in fundamental 
biological processes into a single world-leading unit.

Since it was established in August 2016, LISCB has received significant 
investment from the University and more than £12million in external funding. 
This includes a major investment in a state-of-the-art cryo-electron microscopy 
facility and significant upgrades to the NMR facility.

We are looking to recruit a Research Institute Manager to further develop the 
scientific direction and impact of the LISCB, working closely with the 
Institute Director and other senior staff. The successful candidate will 
develop and maintain the strategic scientific vision of the Institute and 
establish the Institute’s long-term sustainability. They will also engage in 
events and activities to promote the LISCB’s national and international profile 
and reputation.

Please go to https://jobs.le.ac.uk/vacancies/vacancy-details.aspx?VacancyID=332 
for more information and to apply.


Dr. T.J. Ragan

Senior Research Computation Officer
Leicester Institute of Structural and Chemical Biology
University of Leicester, University Road, Leicester LE1 7RH, UK

t: +44 (0)116 223 1287
e: tj.ra...@leicester.ac.uk
w: www.le.ac.uk/liscb

[University of Leicester Logo][Athena Swan Silver Award]







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