Re: [ccp4bb] Phasing with Many Monomers/AU
Dear all, I don't know how closely this relates to what James pointed at, but regarding the aspect of NCS information usage in the heavy-atom finding/refinement stage, I would like to mention two programs of my knowledgethat are able to detect NCS in a set of putative heavy atom sites froma substructure solution trial: Professs (CCP4) and SitCom. Probaby I missed others ... These are useful in particular for cases like heavy atom-soaked structures, where site occupancies are partial and hence peak heights a weak indicator ofcorrectness, but the binding sites are not random i.e. still comply to the NCS. Both programs are based on the identification of matching triangles among HA sites. (meaning that the approach is less suited for cases with less than 3 sites per monomer). SitCom extracts HA site matches, no matter whether (closed) rotational NCS or purely translational NCS is present; and it can try to determine an unknown NCS order or apply an expected one. No matter which program used, the main benefit in my opinion is telling correct sites (NCS-conform) from wrong ones, so that ideally the filtering results in an improved substructure and HA phase set. Kind regards, Fabio Am 1/23/14 1:00 AM, schrieb CCP4BB automatic digest system: Date:Tue, 21 Jan 2014 18:24:34 -0800 From:James Holton jmhol...@lbl.gov Subject: Re: Phasing with Many Monomers/AU [...] but your initial problems are going to be phasing. Ideally what you'd want is a way of folding back NCS information into the heavy atom finding and phase refinement process, but I know of no programs that actually do that. In fact, both molecular replacement and heavy-atom finding are hindered by this pseodo-translation rather than helped by it. Personally, I blame the fact that methods developers seldom get their hands on interesting datasets like yours. [...] -- Dr. rer. nat. Fabio Dall'Antonia European Molecular Biology Laboratory c/o DESY Notkestraße 85, Bldg. 25a D-22603 Hamburg phone: +49 (0)40 89902-178 fax:+49 (0)40 89902-149 e-mail: fabio.dallanto...@embl-hamburg.de
Re: [ccp4bb] Phasing with Many Monomers/AU
Dear James and all,
just to throw into the pot an idea I never came across anywhere,
and I could never test because I never had such data/problem:
1. many NCS copies in the asymmetric unit;
2. either isomorphous differences and/or anomalous differences from heavy atoms
3. more than one heavy atom bound per molecule;
4. clear self rotation function indicating the directions of the NCS axes and
the point group of the NCS
I always thought in this case one should try and average
under the NCS point symmetry the isomorphous and difference Pattersons around
the origin
to boost the intramolecular heavy-atom Patterson vectors.
Has anyone ever had such data and tried that strategy?
Is it implemented in any of the currently available difference
Patterson-solvers/heavy atom finders?
Ciao!
Pietro
Sent from my Desktop
Dr. Pietro Roversi
Oxford University Biochemistry Department - Glycobiology Division
South Parks Road
Oxford OX1 3QU England - UK
Tel. 0044 1865 275339
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of James Holton
[jmhol...@lbl.gov]
Sent: 22 January 2014 02:24
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Phasing with Many Monomers/AU
The problem of many monomers in the ASU is not restricted to
macromolecules. An interesting recent small molecule example is the
structure of L-tryptophan (http://dx.doi.org/10.1107/S0108768112033484)
which, amazingly, was not published until 2012. This is perhaps in part
due to difficulty in accepting 16 monomers in the ASU (they call this
Z=16), which was unprecedented.
As a beamline scientist, I have seen high Z macromolecular crystals on
many occasions, but they almost never get solved. Yes, they don't
diffract well, but neither does anything else in the early stages of a
project. The reason for not solving them seems more psychological than
anything else. The prospect of amplifying the building and refinement
headache by a factor of Z when Z 10 is perhaps too much for an early
term graduate student to bear.
On the other hand, automated building and refinement has come a long
way, and 24-fold NCS is a great restraint if you can get it! In fact,
for virus structures, it has been shown that you can phase the structure
starting with nothing but a crude spherical envelope and lots of density
modification (http://dx.doi.org/10.1107/S0108767391013211).
but your initial problems are going to be phasing. Ideally what you'd
want is a way of folding back NCS information into the heavy atom
finding and phase refinement process, but I know of no programs that
actually do that. In fact, both molecular replacement and heavy-atom
finding are hindered by this pseodo-translation rather than helped by
it. Personally, I blame the fact that methods developers seldom get
their hands on interesting datasets like yours. And if you look in
the PDB there are very few examples of high Z' structures. Ahem.
Best advice I can give is to try the usual approach, but look very
seriously for NCS as early as you can. Then apply building/phasing
packages like shelx{cde}, phenix.autobuild, or the newly-released Crank2.
-James Holton
MAD Scientist
On 1/18/2014 11:18 PM, Felix Frolow wrote:
Francis, It can happened
We have (not yet published) P1 with 24 molecules. When we cut His-tag we get
P1 with 32 molecules.
In our case we believe it is dictated by very strong interaction between two
monomers, and strong interaction between dimers with build a flattish
tetramer. Probably such formations
is more difficult to oaks than globular oligomers.
In this moment I do not recall what we see in solution, I have to check.
Relating to structure solution, P1 is very convenient space group.
I would go for determination this structure by SAD (SHELXC/D/E pipe, PHENIX
or SHARP). For the native - molecular replacement.
In our time after tremendous developments in Refmac and Phenix and
development o DM refinement is 3-3.4 Ang. Is not very difficult.
I would use in addition to NCS restraints in refinement also multi crystal
averaging. Roumors say it is the most strongest phasing method (attributed to
Eleanor Dodson, myself never used it).
FF
Dr Felix Frolow
Professor of Structural Biology and Biotechnology, Department of Molecular
Microbiology and Biotechnology
Tel Aviv University 69978, Israel
Acta Crystallographica F, co-editor
e-mail: mbfro...@post.tau.ac.il
Tel: ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608
On Jan 19, 2014, at 08:48 , Francis Reyes francis.re...@colorado.edu wrote:
You sure about this space group? 24 monomers in P1 is unusual (at least to
me)
F
On Jan 18, 2014, at 9:14 AM, Chris Fage cdf...@gmail.com wrote:
Hello Everyone,
I am currently trying to phase a structure with an asymmetric unit
predicted to contain 20-24 monomers (space group P1). The native crystals,
while beautiful in appearance (see attached), only diffract to ~3.4-3.0
Re: [ccp4bb] Phasing with Many Monomers/AU
The problem of many monomers in the ASU is not restricted to
macromolecules. An interesting recent small molecule example is the
structure of L-tryptophan (http://dx.doi.org/10.1107/S0108768112033484)
which, amazingly, was not published until 2012. This is perhaps in part
due to difficulty in accepting 16 monomers in the ASU (they call this
Z=16), which was unprecedented.
As a beamline scientist, I have seen high Z macromolecular crystals on
many occasions, but they almost never get solved. Yes, they don't
diffract well, but neither does anything else in the early stages of a
project. The reason for not solving them seems more psychological than
anything else. The prospect of amplifying the building and refinement
headache by a factor of Z when Z 10 is perhaps too much for an early
term graduate student to bear.
On the other hand, automated building and refinement has come a long
way, and 24-fold NCS is a great restraint if you can get it! In fact,
for virus structures, it has been shown that you can phase the structure
starting with nothing but a crude spherical envelope and lots of density
modification (http://dx.doi.org/10.1107/S0108767391013211).
but your initial problems are going to be phasing. Ideally what you'd
want is a way of folding back NCS information into the heavy atom
finding and phase refinement process, but I know of no programs that
actually do that. In fact, both molecular replacement and heavy-atom
finding are hindered by this pseodo-translation rather than helped by
it. Personally, I blame the fact that methods developers seldom get
their hands on interesting datasets like yours. And if you look in
the PDB there are very few examples of high Z' structures. Ahem.
Best advice I can give is to try the usual approach, but look very
seriously for NCS as early as you can. Then apply building/phasing
packages like shelx{cde}, phenix.autobuild, or the newly-released Crank2.
-James Holton
MAD Scientist
On 1/18/2014 11:18 PM, Felix Frolow wrote:
Francis, It can happened
We have (not yet published) P1 with 24 molecules. When we cut His-tag we get
P1 with 32 molecules.
In our case we believe it is dictated by very strong interaction between two
monomers, and strong interaction between dimers with build a flattish tetramer.
Probably such formations
is more difficult to oaks than globular oligomers.
In this moment I do not recall what we see in solution, I have to check.
Relating to structure solution, P1 is very convenient space group.
I would go for determination this structure by SAD (SHELXC/D/E pipe, PHENIX or
SHARP). For the native - molecular replacement.
In our time after tremendous developments in Refmac and Phenix and development
o DM refinement is 3-3.4 Ang. Is not very difficult.
I would use in addition to NCS restraints in refinement also multi crystal
averaging. Roumors say it is the most strongest phasing method (attributed to
Eleanor Dodson, myself never used it).
FF
Dr Felix Frolow
Professor of Structural Biology and Biotechnology, Department of Molecular
Microbiology and Biotechnology
Tel Aviv University 69978, Israel
Acta Crystallographica F, co-editor
e-mail: mbfro...@post.tau.ac.il
Tel: ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608
On Jan 19, 2014, at 08:48 , Francis Reyes francis.re...@colorado.edu wrote:
You sure about this space group? 24 monomers in P1 is unusual (at least to me)
F
On Jan 18, 2014, at 9:14 AM, Chris Fage cdf...@gmail.com wrote:
Hello Everyone,
I am currently trying to phase a structure with an asymmetric unit predicted to
contain 20-24 monomers (space group P1). The native crystals, while beautiful
in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and
SeMet-derived crystals grow with poor morphology (small needles). Also, based a
fluorescence scan, I know that mercury does not bind appreciably. Other than
screening for a new space group, what options might I have for phasing this
many monomers at lower resolution? Is there any real chance of solving the
structure in this space group?
Thank you in advance for any suggestions!
Regards,
Chris
Crystals.jpg
Re: [ccp4bb] Phasing with Many Monomers/AU
In my experience translational NCS also can a part when one has many molecules in the a.u. If MR is an option, modern packages are rather good in dealing with TNCS. We used Molrep + Refmac at 3.x A (still unpublished) for a case with 18 complexes (36 monomers) in the a.u. and things weren't as bad as I originally thought. BTW, we later made the construct 3 aa longer and we got 1 dimer in the a.u. with xtals diffracting to 2 A. I would consider playing a bit with your construct (not only the tag). Good luck! R On 18 Jan 2014, at 17:14, Chris Fage cdf...@gmail.commailto:cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris Crystals.jpg Roberto A. Steiner Group Leader Randall Division of Cell and Molecular Biophysics King's College London roberto.stei...@kcl.ac.ukmailto:roberto.stei...@kcl.ac.uk Room 3.10A New Hunt's House Guy's Campus SE1 1UL London Phone 0044 20 78488216 Fax0044 20 78486435
Re: [ccp4bb] Phasing with Many Monomers/AU
Is the monomer the biggest unit you have to search with? If there is a dimer, tetramer, etc. that is conserved, you could try searching with that. On 01/19/14 14:30, Chris Fage wrote: Thank you all for your responses. I already have a few ideas about how to approach the problem. One of my concerns with so monomers per asymmetric unit at lower resolution was the failure of MR software. Neither PHENIX nor Phaser MR have made progress. I am fairly new to anomalous methods, having solved only two structures by SeMet-based SAD. I've certainly picked up on a number of tricks from the recent messages on heavy atoms, but I thought my case might be a little unusual. I am confident the space group is P1, as it was the only viable option when I indexed four clean albeit low-res datasets. The monomers are ~38 kDa, and the crystals diffracted to 3.4-3.0 at a synchrotron. The conditions for both native and SeMet crystals are: 8-12% PEG 2000 MME, 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 5.5. Macromolecular seeding of native crystals into SeMet drops yields the needle-like crystals. Any further input is greatly appreciated! Regards, Chris On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage cdf...@gmail.com mailto:cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Phasing with Many Monomers/AU
I agree. Searching with a larger unit is likely to be successful if you have a good idea of the structure of that larger unit. We had an example of a low homology (29% identity) MR situation with 8 subunits per ASU with twinned data. Not solvable with monomers. Solvable with a dimer search model, then feeding that solution to Parrot and Buccaneer for density modification and automated chain-building using 8-fold NCS. Buccaneer built about 95%+ of the structure correctly. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 1/20/2014 8:41 AM, David Schuller wrote: Is the monomer the biggest unit you have to search with? If there is a dimer, tetramer, etc. that is conserved, you could try searching with that. On 01/19/14 14:30, Chris Fage wrote: Thank you all for your responses. I already have a few ideas about how to approach the problem. One of my concerns with so monomers per asymmetric unit at lower resolution was the failure of MR software. Neither PHENIX nor Phaser MR have made progress. I am fairly new to anomalous methods, having solved only two structures by SeMet-based SAD. I've certainly picked up on a number of tricks from the recent messages on heavy atoms, but I thought my case might be a little unusual. I am confident the space group is P1, as it was the only viable option when I indexed four clean albeit low-res datasets. The monomers are ~38 kDa, and the crystals diffracted to 3.4-3.0 at a synchrotron. The conditions for both native and SeMet crystals are: 8-12% PEG 2000 MME, 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 5.5. Macromolecular seeding of native crystals into SeMet drops yields the needle-like crystals. Any further input is greatly appreciated! Regards, Chris On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage cdf...@gmail.com mailto:cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Phasing with Many Monomers/AU
What is the sequence identity of your best search model? Finding that many copies in P1 with 3A data is a challenge but certainly not impossible if there is a reasonably close (20-25% identity) search model available. I would suggest spending some time on preparing a very good search model with tools like Sculptor as well as manually trimming loops and using ensembles of conserved folds from several homologous structures. It is also worth remembering that there are a number of different programs available for molecular replacement and it is worth investing some time in learning how to use Molrep and Amore as well as Phaser as they all have different strengths and weaknesses. Is your data otherwise devoid of any other problems like pseudo-translational symmetry? These can be readily identified with tools like phenix.xtriage. PST can complicate matters quite considerably in molecular replacement. SAD phases, even if obtained at low-resolution, can still be very useful if combined with molecular replacement, so it is well worth pursuing all lines of attack simultaneously. Hope this helps. Eugene On 19 January 2014 19:30, Chris Fage cdf...@gmail.com wrote: Thank you all for your responses. I already have a few ideas about how to approach the problem. One of my concerns with so monomers per asymmetric unit at lower resolution was the failure of MR software. Neither PHENIX nor Phaser MR have made progress. I am fairly new to anomalous methods, having solved only two structures by SeMet-based SAD. I've certainly picked up on a number of tricks from the recent messages on heavy atoms, but I thought my case might be a little unusual. I am confident the space group is P1, as it was the only viable option when I indexed four clean albeit low-res datasets. The monomers are ~38 kDa, and the crystals diffracted to 3.4-3.0 at a synchrotron. The conditions for both native and SeMet crystals are: 8-12% PEG 2000 MME, 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 5.5. Macromolecular seeding of native crystals into SeMet drops yields the needle-like crystals. Any further input is greatly appreciated! Regards, Chris On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris -- Dr Eugene Valkov Room 3N049 Division of Structural Studies MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. Email: eval...@mrc-lmb.cam.ac.uk Tel: +44 (0) 1223 267358
Re: [ccp4bb] Phasing with Many Monomers/AU
It will be useful if you share the unit cell dimensions, may be it belongs to a higher symmetry, given the low resolution, you may have missed it out. Sridhar From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eugene Valkov Sent: Monday, January 20, 2014 6:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Phasing with Many Monomers/AU What is the sequence identity of your best search model? Finding that many copies in P1 with 3A data is a challenge but certainly not impossible if there is a reasonably close (20-25% identity) search model available. I would suggest spending some time on preparing a very good search model with tools like Sculptor as well as manually trimming loops and using ensembles of conserved folds from several homologous structures. It is also worth remembering that there are a number of different programs available for molecular replacement and it is worth investing some time in learning how to use Molrep and Amore as well as Phaser as they all have different strengths and weaknesses. Is your data otherwise devoid of any other problems like pseudo-translational symmetry? These can be readily identified with tools like phenix.xtriage. PST can complicate matters quite considerably in molecular replacement. SAD phases, even if obtained at low-resolution, can still be very useful if combined with molecular replacement, so it is well worth pursuing all lines of attack simultaneously. Hope this helps. Eugene On 19 January 2014 19:30, Chris Fage cdf...@gmail.com mailto:cdf...@gmail.com wrote: Thank you all for your responses. I already have a few ideas about how to approach the problem. One of my concerns with so monomers per asymmetric unit at lower resolution was the failure of MR software. Neither PHENIX nor Phaser MR have made progress. I am fairly new to anomalous methods, having solved only two structures by SeMet-based SAD. I've certainly picked up on a number of tricks from the recent messages on heavy atoms, but I thought my case might be a little unusual. I am confident the space group is P1, as it was the only viable option when I indexed four clean albeit low-res datasets. The monomers are ~38 kDa, and the crystals diffracted to 3.4-3.0 at a synchrotron. The conditions for both native and SeMet crystals are: 8-12% PEG 2000 MME, 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 5.5. Macromolecular seeding of native crystals into SeMet drops yields the needle-like crystals. Any further input is greatly appreciated! Regards, Chris On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage cdf...@gmail.com mailto:cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris -- Dr Eugene Valkov Room 3N049 Division of Structural Studies MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. Email: eval...@mrc-lmb.cam.ac.uk mailto:eval...@mrc-lmb.cam.ac.uk Tel: +44 (0) 1223 267358
Re: [ccp4bb] Phasing with Many Monomers/AU
Hi, In the past I had two cases where seemingly unsuccessful MR became successful simply by collecting the missing cusp, which is due to exist in your P1 case if you collected your data by rotation of the crystal around a single orientation. However, I don't know if modern MR programs use techniques that overcome that problem. Carlos G. Sridhar Prasad wrote: It will be useful if you share the unit cell dimensions, may be it belongs to a higher symmetry, given the low resolution, you may have missed it out. Sridhar From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eugene Valkov Sent: Monday, January 20, 2014 6:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Phasing with Many Monomers/AU What is the sequence identity of your best search model? Finding that many copies in P1 with 3A data is a challenge but certainly not impossible if there is a reasonably close (20-25% identity) search model available. I would suggest spending some time on preparing a very good search model with tools like Sculptor as well as manually trimming loops and using ensembles of conserved folds from several homologous structures. It is also worth remembering that there are a number of different programs available for molecular replacement and it is worth investing some time in learning how to use Molrep and Amore as well as Phaser as they all have different strengths and weaknesses. Is your data otherwise devoid of any other problems like pseudo-translational symmetry? These can be readily identified with tools like phenix.xtriage. PST can complicate matters quite considerably in molecular replacement. SAD phases, even if obtained at low-resolution, can still be very useful if combined with molecular replacement, so it is well worth pursuing all lines of attack simultaneously. Hope this helps. Eugene On 19 January 2014 19:30, Chris Fage cdf...@gmail.com mailto:cdf...@gmail.com wrote: Thank you all for your responses. I already have a few ideas about how to approach the problem. One of my concerns with so monomers per asymmetric unit at lower resolution was the failure of MR software. Neither PHENIX nor Phaser MR have made progress. I am fairly new to anomalous methods, having solved only two structures by SeMet-based SAD. I've certainly picked up on a number of tricks from the recent messages on heavy atoms, but I thought my case might be a little unusual. I am confident the space group is P1, as it was the only viable option when I indexed four clean albeit low-res datasets. The monomers are ~38 kDa, and the crystals diffracted to 3.4-3.0 at a synchrotron. The conditions for both native and SeMet crystals are: 8-12% PEG 2000 MME, 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 5.5. Macromolecular seeding of native crystals into SeMet drops yields the needle-like crystals. Any further input is greatly appreciated! Regards, Chris On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage cdf...@gmail.com mailto:cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris -- Dr Eugene Valkov Room 3N049 Division of Structural Studies MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. Email: eval...@mrc-lmb.cam.ac.uk mailto:eval...@mrc-lmb.cam.ac.uk Tel: +44 (0) 1223 267358 -- ** Dr. Carlos Frazao Structural Biology Laboratory - Macromolecular Crystallography Unit ITQB-UNL, Av Republica, Apartado 127 2781-901 Oeiras, Portugal Phone: (351)-214469666 FAX:(351)-214433644 e-mail: fra...@itqb.unl.pt www.itqb.unl.pt
Re: [ccp4bb] Phasing with Many Monomers/AU
I am grateful for all of the suggestions. I think I have enough tricks to try at this point, but I may check back with this group if things don't work out. Many thanks once again, Chris On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris
Re: [ccp4bb] Phasing with Many Monomers/AU
Wasn't there this huge thread just 3 days ago on heavy atom soaking On 19/01/2014 07:18, Felix Frolow wrote: Francis, It can happened We have (not yet published) P1 with 24 molecules. When we cut His-tag we get P1 with 32 molecules. In our case we believe it is dictated by very strong interaction between two monomers, and strong interaction between dimers with build a flattish tetramer. Probably such formations is more difficult to oaks than globular oligomers. In this moment I do not recall what we see in solution, I have to check. Relating to structure solution, P1 is very convenient space group. I would go for determination this structure by SAD (SHELXC/D/E pipe, PHENIX or SHARP). For the native - molecular replacement. In our time after tremendous developments in Refmac and Phenix and development o DM refinement is 3-3.4 Ang. Is not very difficult. I would use in addition to NCS restraints in refinement also multi crystal averaging. Roumors say it is the most strongest phasing method (attributed to Eleanor Dodson, myself never used it). FF Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Jan 19, 2014, at 08:48 , Francis Reyes francis.re...@colorado.edu wrote: You sure about this space group? 24 monomers in P1 is unusual (at least to me) F On Jan 18, 2014, at 9:14 AM, Chris Fage cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris Crystals.jpg
Re: [ccp4bb] Phasing with Many Monomers/AU
Thank you all for your responses. I already have a few ideas about how to approach the problem. One of my concerns with so monomers per asymmetric unit at lower resolution was the failure of MR software. Neither PHENIX nor Phaser MR have made progress. I am fairly new to anomalous methods, having solved only two structures by SeMet-based SAD. I've certainly picked up on a number of tricks from the recent messages on heavy atoms, but I thought my case might be a little unusual. I am confident the space group is P1, as it was the only viable option when I indexed four clean albeit low-res datasets. The monomers are ~38 kDa, and the crystals diffracted to 3.4-3.0 at a synchrotron. The conditions for both native and SeMet crystals are: 8-12% PEG 2000 MME, 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 5.5. Macromolecular seeding of native crystals into SeMet drops yields the needle-like crystals. Any further input is greatly appreciated! Regards, Chris On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris
Re: [ccp4bb] Phasing with Many Monomers/AU
Chris, On Jan 19, 2014, at 11:30 AM, Chris Fage cdf...@gmail.com wrote: Thank you all for your responses. I already have a few ideas about how to approach the problem. One of my concerns with so monomers per asymmetric unit at lower resolution was the failure of MR software. Neither PHENIX nor Phaser MR have made progress. I am fairly new to anomalous methods, having solved only two structures by SeMet-based SAD. I've certainly picked up on a number of tricks from the recent messages on heavy atoms, but I thought my case might be a little unusual. I am confident the space group is P1, as it was the only viable option when I indexed four clean albeit low-res datasets. The monomers are ~38 kDa, and the crystals diffracted to 3.4-3.0 at a synchrotron. You'll probably get a lot of (good) suggestions on the ccp4bb, but being new to structure solving by anomalous methods, you should seek out someone who can walk with you through low resolution structure solution. As you may have learned, when working at these resolutions, structures just don't 'pop' out. [advertisement] I'd recommend the Rapidata course at BNL, which meets in the spring. When I last spoke with Bob recently, I had the impression there were open positions. You can bring your data and challenge the experts (many of whom are directly involved in the crystallography software development). http://www.bnl.gov/RapiData/ Disclaimer: I am not affiliated with the course :) Just an alum. [/advertisement] Cheers, F The conditions for both native and SeMet crystals are: 8-12% PEG 2000 MME, 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 5.5. Macromolecular seeding of native crystals into SeMet drops yields the needle-like crystals. Any further input is greatly appreciated! Regards, Chris On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris
[ccp4bb] Phasing with Many Monomers/AU
Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris attachment: Crystals.jpg
Re: [ccp4bb] Phasing with Many Monomers/AU
Hi Chris, It would be nice to have a wee bit more information. Is 3.4-3.0 angstroms from a home source or synchrotron? What are the crystallization conditions for both the native and SeMet crystals? Did you see the SeMet crystals with the native crystals. Have you tried MMS with the native crystals into new screening conditions. Cheers, Scott On Jan 18, 2014, at 12:14 PM, Chris Fage cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris Crystals.jpg
Re: [ccp4bb] Phasing with Many Monomers/AU
You sure about this space group? 24 monomers in P1 is unusual (at least to me) F On Jan 18, 2014, at 9:14 AM, Chris Fage cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris Crystals.jpg
Re: [ccp4bb] Phasing with Many Monomers/AU
Francis, It can happened We have (not yet published) P1 with 24 molecules. When we cut His-tag we get P1 with 32 molecules. In our case we believe it is dictated by very strong interaction between two monomers, and strong interaction between dimers with build a flattish tetramer. Probably such formations is more difficult to oaks than globular oligomers. In this moment I do not recall what we see in solution, I have to check. Relating to structure solution, P1 is very convenient space group. I would go for determination this structure by SAD (SHELXC/D/E pipe, PHENIX or SHARP). For the native - molecular replacement. In our time after tremendous developments in Refmac and Phenix and development o DM refinement is 3-3.4 Ang. Is not very difficult. I would use in addition to NCS restraints in refinement also multi crystal averaging. Roumors say it is the most strongest phasing method (attributed to Eleanor Dodson, myself never used it). FF Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Jan 19, 2014, at 08:48 , Francis Reyes francis.re...@colorado.edu wrote: You sure about this space group? 24 monomers in P1 is unusual (at least to me) F On Jan 18, 2014, at 9:14 AM, Chris Fage cdf...@gmail.com wrote: Hello Everyone, I am currently trying to phase a structure with an asymmetric unit predicted to contain 20-24 monomers (space group P1). The native crystals, while beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms at best, and SeMet-derived crystals grow with poor morphology (small needles). Also, based a fluorescence scan, I know that mercury does not bind appreciably. Other than screening for a new space group, what options might I have for phasing this many monomers at lower resolution? Is there any real chance of solving the structure in this space group? Thank you in advance for any suggestions! Regards, Chris Crystals.jpg
