All:
While Phil Jeffrey attributed to me the "trick" of aligning the hinge axis of
an Fab along the Z direction, I, in turn, must give credit to Mirek Cygler, who
explained this to me at the Diffraction Methods in Molecular Biology [now
Structural Biology] Gordon Research Conference in 1986.
To Scott's query about searching for "1 domain sequentially and then other
domains OR searching for multiple domains all at once?"
Inevitably a program like PHASER searches for domains ("ensembles" in its
terminology) sequentially. However, as to the practical question of whether to
"feed" PHASER all of the domains in one run, that is certainly how I start and
it is usually (almost always?) successful. In fact, while I no longer bother to
align the hinge axis of an Fab along the Z axis, I now break Fabs into three
parts: CL:CH1, VH, and VL to allow molecular replacement to accommodate the
"tilt" angle between VH and VL (tilt angle is a term I learned from Gary
Gilliland's talk at the Diffraction Methods in Structural Biology GRC in 2014).
This also allows me to search for the highest identity VL and, separately, VH
in the PDB to use as probe models. N.B. since I'm usually studying antigen/Fab
complex, I'm usually searching for 4 ensembles in one PHASER run: CL:CH1,
antigen, VH and VL.
Steven
P.S. to Tassos Perrakis: I'm sorry that these plugs for the Diffraction Methods
GRC are ~4 months too late!
P.P.S. to Eddie Snell: And I'm sorry that these plugs are ~18 months too early
for 2016's Diffraction Methods GRC!
==========
Date: Mon, 6 Oct 2014 18:33:26 +0000
From: Scott Thomas Walsh <swals...@umd.edu<mailto:swals...@umd.edu>>
Subject: Re: Molecular Replacement model preparation
Hi Phil,
Thank you for the input. I would like to get CCP4ers input. I deal with
multiple domain cytokine receptors in a manner very similar to antibody
molecules.
Have people have more correct solutions searching for 1 domain sequentially and
then other domains OR searching for multiple domains all at once? I am curious
to hear peoples' experiences on this topic?
Cheers,
Scott
************************************************
Scott T. R. Walsh, PhD
Assistant Professor
University of Maryland
IBBR/CBMG
3127E CARB-2
9600 Gudelsky Drive
Rockville, MD 20850 USA
phone: (240) 314-6478
fax: (240) 314-6225
email: swals...@umd.edu<mailto:swals...@umd.edu>
On Oct 6, 2014, at 2:11 PM, Phil Jeffrey
<pjeff...@princeton.edu<mailto:pjeff...@princeton.edu>> wrote:
> That document is fairly old and is in dire need of revision to reflect the
> modern arsenal of programs.
>
> Nevertheless:
> Putting the hinge axis along Z was a trick told to me by Steven Sheriff back
> in the days when we worked on Fab structures - which after all are classical
> examples of hinged molecules. One would search with separate domain
> fragments - split either side of the hinge - and the Z-orientation trick
> makes it easier to spot pairs of peaks from each search model that are
> related to each other. In the Fab world we searched with Fv models (VH:VL
> heterodimer) and CH1:CL constant region heterodimeric models. Peaks related
> solely by hinge motion would have similar alpha and beta angles and
> potentially different gamma (Crowther convention Eulerian angles).
> Historical note: this was back in the days when it was possible to remember
> the names of all the Fab fragments that were in PDB and their respective IDs.
>
> This ploy was more important in the days before Phaser or Molrep, which will
> now gleefully try a long list of rotation function peaks for you quite
> quickly, so manually parsing the list of rotation function peaks is rather
> unnecessary. And perhaps counter-productive.
>
>
> Split your molecule apart at the hinge, giving fragment1 and fragment2.
> Attempt to find both fragments independently. Choose the one that gives the
> best results: TFZ score or LLG score or discrimination between possible space
> group or whatever you like. Then, attempt to find the *other* fragment in
> the context of that first solution.
>
>
> Phil Jeffrey
> Princeton
>
>
> On 10/5/14 3:34 AM, Luzuokun wrote:
>> Dear all,
>> I'm doing molecular replacement using Phaser. My protein is predicted
>> to have two domain with a "hinge" linking them. The model sequence
>> identity is 0.27. But the MR result is poor. I've tried other
>> programme (Molrep, MrBump, Balbes,,,_.) But no improvement was
>> observed. I think that this is due to the "open" or "closed"
>> conformation around the hinge. I was told that I could place the Z
>> axis along the hinge
>> (http://xray0.princeton.edu/~phil/Facility/Guides/MolecularReplacemen
>> t.html), could anyone tell me more details about how to do next?
>>
>> Thanks!
>> Lu Zuokun
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