Re: [ccp4bb] Difficult experimental phasing

[Diana Tomchick]

Estimating isomorphism by eyeballing the difference in unit cell lengths may 
mask significant non-isomorphism that is present. Iodine is a large atom and it 
is notoriously difficult to get an iodine derivative that is isomorphous to a 
native protein.

Depending upon the chemistry of your mother liquor, it is very possible that 
you no longer have a significant amount of I3C around, so not seeing a triangle 
of iodines does not mean that your data is worthless.

Instead, I would try MR-SAD again with Phaser, and try on individual and merged 
datasets. If you are merging datasets from multiple crystals, this might be 
problematic if the level of heavy atom substitution is different between 
crystals. You can also try as input the heavy atom solution from HySS or SHELX. 
Try to limit the resolution of your heavy atom search to the resolution of 
measurability for the anomalous signal.

Iodine soaks, especially quick soaks, might not be the best HA derivative in 
your particular case. Try tantalum bromide, that cluster is less likely to 
degrade and will give a stronger signal at lower resolution.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Apr 30, 2018, at 11:28 AM, Abris Bendes  wrote:

Dear all,

I’m trying to solve the structure of a putative homodimer, but despite having 
collected good quality data, it has proven to be rather difficult.

I have collected native and derivative datasets (2.2-4Å, C2 SG, 
165/35/115Å//104° unit cell) with acceptable merging statistics. Xtriage, 
Aimless does not report any apparent twinning, tNCS or significant anisotropy. 
Based on Matthews coefficient, two monomers should be present in the ASU. 
Self-rotation function doesn’t give a clear idea on NCS, I see a few weak peaks 
at Chi=180°. Crystal was checked against contaminant with SDS-PAGE, MS and 
ContaMiner.

MR approaches so far have failed due to low sequence homology (~20%). I have 
tried MoRDa, MrBUMP, Balbes or ensembles of different models as homodimer, 
monomer or divided into smaller domains. I can place the dimer or a monomer 
with high TFZ score (6-30), but with smaller domains MR usually thrashes. The 
solution always has a rather high R factor (over 0.5), unclear electron density 
and does not improve on subsequent intensive rebuilding. Trials in P1 (or in 
other possible spacegroups) have failed too.

I have recently collected multiple good datasets from a single I3C soaked 
crystal with measurability extending to 4-7Å on individual datasets or 3-4Å on 
merged multi-dataset data. No apparent radiation damage or other pathologies 
present. Moderate isomorphism with native data (<0.4% unit cell difference).

Any attempt to solve the substructure or get interpretable maps has so far 
failed even with exhaustive SHELX, CRANK2, HySS, Autosol, Phaser or 
AutoRickshaw runs. MR-SAD or SIRAS approaches have so far failed too. Despite 
promising HySS solutions (CC>0.35) I can´t see the expected triangle 
arrangement of the I atoms, can’t discern the hands and it always result in 
uninterpretable low FOM maps.

I have a few weaker datasets (not as isomorphous though) with different HA 
soaks (signal up to 7Å), so I could try MIR or multi-crystal averaging, though 
I have no experience with these approaches.

I can provide more details if needed and I would appreciate any kind of help 
with my problem.

Thank you very much.

Best regards,
Abris




UT Southwestern


Medical Center



The future of medicine, today.

[ccp4bb] Acrimboldo BORGES standard libraries

[Jan Abendroth]

Hi all,
I have been trying to install the BORGES standard libraries to run
ACRIMBOLDO with them.

This is an installation of the libraries on top of a CCP4 installation
using 'ccp4sm'. The libraries download, the installation finishes and the
libraries end up in  the following folder:
$CCP4/bin/destination/BORGES_LIBS
I selected 'edit set up file' while running 'ccp4sm'. Still, the standard
libraries show in ccp4i remain greyed out, only CUSTOM is an option. The
ccp4.setup-sh file does not seem to have a reference to BORGES.

I wonder what I am missing to get this installed.

Thanks,
Jan

-- 
Jan Abendroth
Emerald Biostructures
Seattle / Bainbridge Island, WA, USA
home: Jan.Abendroth_at_gmail.com
work: JAbendroth_at_embios.com
http://www.emeraldbiostructures.com

[ccp4bb] Difficult experimental phasing

[Abris Bendes]

Dear all,

I’m trying to solve the structure of a putative homodimer, but despite having 
collected good quality data, it has proven to be rather difficult.

I have collected native and derivative datasets (2.2-4Å, C2 SG, 
165/35/115Å//104° unit cell) with acceptable merging statistics. Xtriage, 
Aimless does not report any apparent twinning, tNCS or significant anisotropy. 
Based on Matthews coefficient, two monomers should be present in the ASU. 
Self-rotation function doesn’t give a clear idea on NCS, I see a few weak peaks 
at Chi=180°. Crystal was checked against contaminant with SDS-PAGE, MS and 
ContaMiner.

MR approaches so far have failed due to low sequence homology (~20%). I have 
tried MoRDa, MrBUMP, Balbes or ensembles of different models as homodimer, 
monomer or divided into smaller domains. I can place the dimer or a monomer 
with high TFZ score (6-30), but with smaller domains MR usually thrashes. The 
solution always has a rather high R factor (over 0.5), unclear electron density 
and does not improve on subsequent intensive rebuilding. Trials in P1 (or in 
other possible spacegroups) have failed too.

I have recently collected multiple good datasets from a single I3C soaked 
crystal with measurability extending to 4-7Å on individual datasets or 3-4Å on 
merged multi-dataset data. No apparent radiation damage or other pathologies 
present. Moderate isomorphism with native data (<0.4% unit cell difference).

Any attempt to solve the substructure or get interpretable maps has so far 
failed even with exhaustive SHELX, CRANK2, HySS, Autosol, Phaser or 
AutoRickshaw runs. MR-SAD or SIRAS approaches have so far failed too. Despite 
promising HySS solutions (CC>0.35) I can´t see the expected triangle 
arrangement of the I atoms, can’t discern the hands and it always result in 
uninterpretable low FOM maps.

I have a few weaker datasets (not as isomorphous though) with different HA 
soaks (signal up to 7Å), so I could try MIR or multi-crystal averaging, though 
I have no experience with these approaches.

I can provide more details if needed and I would appreciate any kind of help 
with my problem.

Thank you very much.

Best regards,
Abris

Re: [ccp4bb] Suggestions on Dynamic Light Scattering instruments

[Debasish Kumar Ghosh]

Dear Chandramohan,

We use Malvern particle size analyzer (ZEN 3690 ZETASIZER NANOZS 90, version 
7.03) fitted with 21CFR part 11 software. It works fine for us in terms of 
measuring soluble proteins particles in size range from nanometre to micrometre.

Hope this helps.

Cheers!!

Debasish

__

Debasish Kumar Ghosh

CSIR- Senior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html



- Original Message -
From: Chandramohan Kattamuri <1c5b7cb6c764-dmarc-requ...@jiscmail.ac.uk>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Mon, 30 Apr 2018 20:58:33 +0530 (IST)
Subject: [ccp4bb] Suggestions on Dynamic Light Scattering instruments

Dear all,

We are looking for Dynamic Light Scattering instruments to test our purified 
protein complexes prior to crystallization. Any suggestions or recommendations 
regarding which instruments to pursue?

Thanks in advance


Chandra

kattamuricha...@yahoo.com

[ccp4bb] Suggestions on Dynamic Light Scattering instruments

[Chandramohan Kattamuri]

Dear all,

We are looking for Dynamic Light Scattering instruments to test our purified 
protein complexes prior to crystallization. Any suggestions or recommendations 
regarding which instruments to pursue?

Thanks in advance


Chandra

kattamuricha...@yahoo.com

Re: [ccp4bb] size exclusion columns

[Mahey, Jas]

Dear Markus

Please find a response from one of our specialists - feel free to contact him 
for more information (his contact details are at the bottom of this email as he 
is not on the bulletin board).

The primary factors I would consider are the type of material in the column and 
also the pore size/ effective molecular weight range. The pore size will 
dictate the selectivity of the separation, you mention you are looking at large 
proteins (MDa) so a column with larger pores will give you a higher effective 
molecular weight range and better resolution. The molecular weight range for 
each column is published by each manufacturer, I have listed these below: 

SEPAX UNIX-C 5,000-1,250,000 (https://sepax-tech.com/Unix-C.php) 
BioZen SEC-3 10,000-700,000 (http://www.phenomenex.com/products/detail/biozen) 
Waters BEH450 or XBridge450  SEC 100,000-1,500,000 
(http://www.waters.com/waters/partDetail.htm?partNumber=186006852) 

The SEPAX and BioZen are both silica materials so high efficiency and good 
pressure tolerance but also with the potential for proteins to stick requiring 
high salt. The Xbridge and BEH are hybrid particles (hybrid of a silica and 
polymer) giving reduced secondary interactions but retaining the high 
efficiency and pressure tolerance of silica. so may be a nice middle ground 
between silica and polymer.   

I was also wondering if you working with a HPLC system or a UPLC system? The 
extra column volume has a big impact on resolution of peaks in SEC if you are 
working on HPLC 7.8mm i.d. column will help to absorb some of the system 
dispersion, if your system is a well optimised UPLC (1290, ACQUITY etc) 4.6mm 
will be fine. 

Richard Robinson 
Biopharm Specialist
UK and Ireland  

[T] +442082386100   
[M] +447824326822   
[W] www.waters.com 
[E] richard_robin...@waters.com 


-Original Message-
From: CCP4 bulletin board  On Behalf Of Markus Heckmann
Sent: 26 April 2018 17:04
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] size exclusion columns

Dear all,

We are looking for a size exclusion chromatography column
(silica-based) for protein purification prior to a MALS-detector. We looked for 
(www.waters.com BEH-450), Sepax (Unix-C 300) and Phenomex (BioZen SEC-3).  Any 
'column' tips or recommendations when dealing with large proteins (MDa)?

Many thanks
Markus

[ccp4bb] Paul Scherrer Institut (PSI) :: Scientist

[Schertler Gebhard (PSI)]

https://www.psi.ch/pa/job-opportunities/1753




Prof. Gebhard F.X. Schertler
Structural Biology ETH Zürich D-BIOL

Head of Biology and Chemistry
Division
Paul Scherrer Institut
Laboratory of Biomolecular Research,
LBR
OFLC 109
CH-5232 Villigen PSI
gebhard.schert...@psi.ch
phone +41 56 310 4265

[ccp4bb] Two Postdoctoral Research Posts in Oxford

[Susan Lea]

https://www.path.ox.ac.uk/content/postdoctoral-research-assistant-structural-biologyprotein-chemistry-2-posts

POSTDOCTORAL RESEARCH ASSISTANT IN STRUCTURAL BIOLOGY/PROTEIN CHEMISTRY (2 
POSTS)

Closing date: Friday, 11 May 2018 - 12pm
Grade 7: £31,604 - £38,833 p.a.

We are seeking to appoint two talented Postdoctoral Research Assistants under 
the supervision of Professor Susan Lea 
(https://www.path.ox.ac.uk/content/susan-lea). This project aims to dissect 
innate immune system regulatory pathways and how these are manipulated by 
bacterial and parasite pathogens. These posts will build on recent work 
identifying inhibitors of complement in tick saliva that allow dissection of 
regulatory pathways in human complement.

The postholder will manage his/her own academic research and administrative 
activities, test hypotheses and analyse scientific data from a variety of 
sources as well as assist with the design and implementation of experiments. 
You will collaborate in the preparation of scientific reports and journal 
articles and the presentation of papers and posters at conferences and also 
represent the research group at external meetings/seminars.

Applicants should have a PhD or equivalent experience/qualification. You should 
have experience in expression and purification of proteins from heterologous 
expression systems and analysis of these proteins using biophysical and 
biochemical techniques is essential together as is an interest in using 
structures to understand biological questions. The postholders must be 
meticulous in their approach to record keeping and highly organised and have 
excellent communication skills. Previous laboratory experience is essential and 
should include a high level of competence in molecular biology techniques (e.g. 
DNA purification, plasmid manipulation).

Both posts are fixed-term appointments available for 2 years. If you are 
interested in this role, and have the skills and experience we are looking for, 
please apply online. You will be required to upload a CV and supporting 
statement as part of your online application.

The closing date for applications is 12.00 midday on Friday 11 May 2018. 
Interviews will be held as soon as possible thereafter.



Susan

Prof. Susan M. Lea,  FMedSci  tel: +44 1865 275181
--
Director of the Central Oxford Structural Microscopy and Imaging Centre & 
Professor of Microbiology
Sir William Dunn School of Pathology, Oxford OX1 3RE Professorial Fellow @ 
WadhamCollege

Re: [ccp4bb] Calculating sigma value

[Ian Tickle]

Dale,

On 19 April 2018 at 17:36, Dale Tronrud  wrote:

>The meaning of the term "e/A^3" as used in Coot has nothing to do
> with the charge of an electron.  The intention of its authors is to
> indicate that the value being represented by the map is the density of
> electrons.  It is the number of electrons per cubic Anstrom at that
> point in space.
>

My intention was certainly not to imply that the unit designated 'e' in the
units of electron density used by Coot should be taken to mean charge (in
fact quite the opposite!), even though 'e' would normally be interpreted
here as the atomic unit of charge (see
https://nvlpubs.nist.gov/nistpubs/Legacy/SP/nistspecialpublication811e2008.pdf,
Table 8).

   I, generally, find it useless to say that a number is a density
> (units of "per volume") without saying density of what.  This is not a
> density of charge, not a density of mass, not a density of rabbits.  It
> is a density of electrons.
>

First, note that (SI Guide cited above, section 7), quote: "The value of a
quantity is its magnitude expressed as the product of a number and a unit,
and the number multiplying the unit is the numerical value of the quantity
expressed in that unit".  So by stating that the electron density is X
electrons/A^3, you are claiming that the number in the above recipe is X
and that the unit is 'electrons/A^3', correct?  I just want to be sure we
are talking about the same thing when referring to a 'unit'.

There are numerous reasons why this terminology is inconsistent,
semantically illogical, unconventional or impermissible under the
universally accepted SI system of units (Système international d'unités),
as well as possibly unlawful in the US!  For starters, the object
'electron' to which we are referring here is already very clear from the
definition, so it's quite unnecessary to repeat that object as a component
of its units.  Here is the definition of 'electron density':

"Electron density is the volume number density of electrons, or the number
of electrons per unit volume".  For the definition of 'number density' see:
https://en.wikipedia.org/wiki/Number_density, quote: "Volume number density
is the number of specified objects per unit volume
: [image: n={\frac {N}{V}},] where *N* is
the total number of objects in a volume *V*.  In SI
 units, number
density is measured in m−3, although cm−3 is often used."

There is of course also line number density and area number density, such
as population density, i.e. the number of people per unit area, or area
number density of people.  See
https://en.wikipedia.org/wiki/Population_density, quote: "Therefore, the
worldwide human population density is around 7,500,000,000 ÷ 510,000,000 =
14.7 per km2 (38 per sq. mi). If only the Earth's land area of 150,000,000
km2 (58,000,000 sq. mi.) is taken into account, then human population
density increases to 50 per km2 (129 per sq. mile)." (note that the unit
'sq. mi' is not permitted under SI !).  Seems pretty clear from this what
the units of area number density are, and by analogy the units of volume
number density.

There doesn't seem to be any ambiguity here that what is being described is
the number density of people, and for anyone who doesn't think Wikipedia
has got it right, for an 'official' example of usage of number density,
please refer to section 6.2.6 in the SI Guide cited above, quote:  "6.2.6
... Example: the number density of Pb atoms is 5 ×106 /m3 but not: the
number density of Pb atoms is 5 M/m3."
Furthermore, are you saying that, for example, the statement "the mass of
an electron is 9.10938356(11)×10^−31 kg" is not sufficiently clear and that
it should be "9.10938356(11)×10^−31 kg/electron" instead?  That would
follow logically from your claim above.  The problem with this is that this
usage is clearly not permitted under the SI system, to which all modern
scientific institutions and journals are signed up to using, without
exception.  Please see the above-mentioned SI Guide document, under 'Check
List for Reviewing Manuscripts', item 6: "Information is not mixed with
unit symbols (or names). For example, the form 'the water content is 20
mL/kg' is used and not '20 mL H2O/kg' or '20 mL of water/kg.' (See Sec.
7.5.)."  So mixing the object 'electron' with the other units (A^-3 or kg)
is absolutely not permissible in SI.  Note that an 'electron' is like 'H2O'
in the sense that both are instances of the class 'Matter', and so neither
can possibly be units (i.e. instances of the class 'Units').  This makes
perfect sense for several reasons:

First, the identical unit applied to different objects is 'commensurable',
i.e. it can be compared, subtracted or added.  So if the number density of
electrons is 5 A^-3 and the number density of protons is 10 A^-3 we can
just add number densities and get the total number density of particles or
matter 

Re: [ccp4bb] determining the point group and the space group

[Thomas White]

Hi Gihan, and anyone else who finds this interesting.  Sorry for the
late response:

> how should one determine a point group and the space group of an
> unknown crystal?
> 
> I have a protein crystal with know unit-cell parameters. (these are
> XFEL data so indexing wouldn't give the point and space groups). I
> checked the PDB, but no luck the PDB structures have the different
> space group assigned, no definitive answer hopefully, somebody can
> point me in the right direction 

Space group determination using serial crystallography data is
different to rotation crystallography because you have to start from
the highest possible symmetry and work downwards by finding and
resolving ambiguities, instead of merging in the lowest possible
symmetry and working upwards by looking for possible symmetries.

Just as with any data, the golden rule is that the space group is
only a hypothesis until the structure is solved (and even then...).

Here's a very brief step by step guide.  Start by determining the cell
parameters, which you've done already.

Say the cell parameters look like a hexagonal P lattice.  Proceed for a
while on the assumption that it really is hexagonal P, but keep the
golden rule in mind.  In this case it might be, amongst others,
monoclinic with two axes similar in length and an angle close to 120
degrees.  Use your crystallographic knowledge to spot centering
possibilities, for example a cubic F lattice might look rhombohedral
with angles of 60 degrees (however, the indexing program should spot
these for you).

Merge the snapshots according to the highest point symmetry permissible
by the lattice.  You can look this up in many places including the
symmetry chart distributed with CrystFEL:
https://www.desy.de/~twhite/crystfel/twin-calculator.pdf
It's always the point group with a grey background in the bottom left
corner of the individual table for the lattice type.  For the example
hexagonal P lattice, it's point group 622.

Do the standard tests on the data, particularly twinning tests
including an L-test.  Whenever you see apparently twinned data with
serial crystallography, either the crystals are physically twinned or
the true symmetry is lower and you need to resolve an indexing
ambiguity and merge again.  You will need to try rounds of ambiguity
resolution until the tests are clean and the structure can be solved.

The difficult part is finding your way through the maze of possible
symmetries.  You can attempt a resolution into any subgroup of the
current symmetry, provided that the "ambiguity operator" is just a
rotation (no reflections/inversions).  The CrystFEL table shows the most
obvious subgroups (the ones with the same lattice type).  The
comprehensive map of possibilities can be found in International Tables
A, Fig 10.1.3.2 (in 5th edition).  There are many special cases, and the
find_ambi tool from the CrystFEL extra programs repository can help you
find cases of "accidental" ambiguities due to the particular values of
the lattice parameters:
https://www.desy.de/~twhite/crystfel/programs.html

If there are signs of twinning, try resolving the ambiguity into each
of the subgroups.  If the ambiguity resolves nicely (one way to tell is
by the correlation coefficient graph, which should separate nicely:
http://journals.iucr.org/j/issues/2016/02/00/zd5001/zd5001fig3.html),
try merging the reindexed patterns in the lower symmetry point group,
and check the twinning tests again.

Once you have a merged set of reflections with no apparent twinning,
things are the same as rotation crystallography.  Examine the systematic
absences to see if they suggest any screw axes.  Try molecular
replacement in all the possible space groups.  Consider revisiting the
earlier steps if there are problems.

It's not easy, but it's also not that difficult, just different to
usual.  I would almost go as far as saying it's fun, like cracking a
secret code.  This kind of situation is my personal favourite part of
crystallography (!)

This recent paper embodies a similar workflow in a nice algorithm:
https://journals.iucr.org/d/issues/2018/05/00/rr5155/

Hope that helps,

Tom

-- 
Thomas White  
4E1F C14D 0E0A A014 FE5D 3FC6 C628 75D1 D4CA 4C30
Direct telephone: +49 (0)40 8998-5786