Dear Scott,
By “AND” searching, presumably you mean adding another SEARCH command? (Or clicking “Add another search” in the ccp4i interface.) For Steven’s strategy, you want to specify separate searches for separate ensembles, i.e. give several SEARCH commands in a script or add extra searches in ccp4i.
The “OR” option (to specify several ensembles to search for in one SEARCH command, available through ccp4i by opening the “Additional Search parameters” pane and turning on “Allow search with alternative ensembles...” uses alternative models to search for the same component, which can be useful but isn’t what you’re interested in for defining a hinge angle.
At some point in the distant past, you had to tell Phaser what order to search for the components in, but then we implemented a method to automatically choose a good search order and, more recently, implemented an improved method to choose the optimal search order. If the information you give Phaser about the quality of the model (RMSD or sequence identity, which Phaser turns into an RMSD estimate) is correct, then the strength of the signal for different models can be estimated. Phaser will use this to define an initial search order. If each search gives an unambiguous solution, then everything will proceed in the predefined order. However, whenever there is ambiguity about whether a correct solution has been found, then Phaser will automatically try strategies like choosing a different search order or changing the resolution of the data used for the calculation.
So, in most cases, it’s best to tell Phaser everything you’re looking for and set up one job to find everything, because this will allow the greatest level of optimisation and the most flexibility in trying different strategies. It’s only if this fails that you’ll want to choose the strategy and parameters manually.
Best wishes,
Randy Read
-----
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 Road E-mail: [log in to unmask]
Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
On 7 Oct 2014, at 15:16, Scott Thomas Walsh <[log in to unmask]> wrote:
> Hi Steven,
>
> Thank you for the information and guidance. When you search for all the ensembles with Phaser do you use “AND” or “OR”
> searching?
>
> Cheers,
>
> Scott
>
>>
>> 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.
>>
>
>
>
> ************************************************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: [log in to unmask]
>
>
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