Hi Carlos,
In a practical setting you don't have to be very purist. The memory with respect to the reflection data is lost if you refine to convergence. Now there was are recent discussion on refinement convergence and again you can be quite purist here. However, if you go through a few cycles of rebuilding and refinement until R and R-free are stable, you are in clear with respect to cheating.*
HTH,
Robbie
When working with ligands there a much more severe ways of cheating (oneself).
> -----Original Message-----
> From: CCP4 bulletin board <[log in to unmask]> On Behalf Of Carlos
> Kikuti
> Sent: Thursday, February 8, 2024 00:24
> To: [log in to unmask]
> Subject: Re: [ccp4bb] what is isomorphous?
>
> Hello!
>
> I have to admit my maths is a bit lazy, but this discussion got me stitched up,
> because of a point I believe has not been addressed: the Rfree flags. I've been
> trained to import Rfree flags whenever the crystals have the same space group
> and similar cell dimensions to the search model for molecular replacement -
> this to avoid "cheating" the Rfree validation with reflections that the search
> model has already 'seen'. We often work with series of crystals of the same
> proteins with different ligands, which give groups of very similar unit cells. So
> far my strategy has been to mirror the Rfree flags (using the -ref or -rfree
> keyword in Autoproc) whenever the biggest difference in one of the dimensions
> is 5% - number just out of my instinct, taking in account the Rmerge of ~0.1 or
> less in the good cases. Maximum resolutions are between 2.7 and 1.9
> angstroms. Now considering the fact that isomorphism depends on resolution,
> it makes me reconsider the 5% cut-off: this might be fine at the low resolution,
> but what about the higher resolution shells? What would be the best way to
> proceed in these cases, then? Because the level of 'cheating' will also vary with
> resolution...
>
> Carlos
>
> On Sun, Dec 31, 2023 at 5:21 PM James Holton <[log in to unmask]
> <mailto:[log in to unmask]> > wrote:
>
>
>
> Ahh yes. I still have the very helpful email I got from Dame Louise
> Johnson in 2010. I don't think she would mind my quoting it here:
>
>
>
> Dear James
>
> I was sorry to miss you when you were at Diamond - I was in
> Germany.
>
> The story of the two forms of lysozyme crystals goes back to
> about 1964 when
> it was found that the diffraction patterns from different crystals
> could be
> placed in one of two classes depending on their intensities.
> This discovery
> was a big set back at the time and I can remember a lecture
> title being
> changed from the 'The structure of lysozyme' to 'The structure
> of lysozyme
> two steps forward and one step back'. Thereafter the crystals
> were
> screened based on intensities of the (11,11,l) rows to
> distinguish them
> (e.g. 11,11,4 > 11,11,5 in one form and vice versa in another).
> Data were
> collected only for those that fulfilled the Type II criteria. (These
> reflections were easy to measure on the linear diffractometer
> because
> crystals were mounted to rotate about the diagonal axis). As I
> recall both
> Type I and Type II could be found in the same crystallisation
> batch .
> Although sometimes the external morphology allowed
> recognition this was not
> infallible.
>
> The structure was based on Type II crystals. Later a graduate
> student Helen
> Handoll examined Type I. The work, which was in the early days
> and before
> refinement programmes, seemed to suggest that the
> differences lay in the
> arrangement of water or chloride molecules (Lysozyme was
> crystallised from
> NaCl). But the work was never written up. Keith Wilson at one
> stage was
> following this up as lysozyme was being used to test data
> collection
> strategies but I do not know the outcome.
>
> An account of this is given in International Table Volume F
> (Rossmann and
> Arnold edited 2001) p760.
>
> Tony North was much involved in sorting this out and if you
> wanted more info
> he would be the person to contact.
>
> I hope this is helpful. Do let me know if you need more.
>
> Best wishes
>
> Louise
>
> Armed with this advice, I searched the PDB using what I call the
> "Johnson ratio" of F(11,11,4) / F(11,11,5) and found there was a continuous
> spectrum (pasted below). The extrema of this spectrum were 3aw6 and 3aw7
> (circled), which are not only from the same paper, but from the same crystal: a
> dehydration study. Despite a modest unit cell size change of 0.7%, the R-factor
> between the Fobs of these two entries (aka R-iso) is 44%. Its like they are
> different proteins, and a 12% change in relative humidity was all it took. I never
> did get a chance to tell Louise that it was a dehydration effect. It took me too
> long to figure it out. But, I expect she would have found that information
> delightful.
>
> To weigh in on the OP:
> First: @Doeke, no I am not reviewing your new paper, but I hope
> whomever is is being helpful.
>
> Second: I am with Randy Read that isomorphism means "same shape",
> and also with Bernhard Rupp that "same" is resolution dependent. Anything is
> "isomorphous" if you stand far enough away from it (like Carl Sagan's "pale blue
> dot"). So, I personally define "isomorphism" in terms of the agreement
> between the structure factors (Fobs). When does it become non-isomorphism? I
> say this is when the changes in Fobs become intolerable. What is intolerable?
> Depends on what you are doing, but in general it is good to compare the effect
> of interest to the existing noise. If the changes in Fobs due to the structural shift
> become larger than SIGFobs, then you start having "non-isomorphism". For the
> common example of merging data from multiple crystals, non-isomorphism
> becomes intolerable when it is large enough to degrade rather than improve
> your signal-to-noise after merging.
>
> For comparing maps, I'd say non-isomorphism becomes intolerable
> when the difference peaks due to uninteresting movements becomes larger
> than those due to interesting changes. What is interesting? Depends on what is
> causing it. Large-scale domain motions due to laser-induced heat are perhaps
> "not interesting" (to some), but large-scale domain motions due to allosteric
> regulation are "interesting" (to some). Other "interesting" things like ligand
> binding are an occupancy shift, which are traditionally not considered non-
> isomorphism because the xyz positions aren't changing (recall the definition of
> "isomorphous replacement"). The term "non-isomorphism" is usually used to
> describe a large-scale positional shift.
>
> These large-scale shifts are perhaps why changes in the unit cell can be
> an indicator of isomorphism, but in my experience this relationship is weak.
> This is especially true with serial crystallography where all three cell dimensions
> are seldom constrained by a single image. That is, there are sources of error
> that affect the accuracy of spot positions (measured cell), but not the intensities
> (structure factors). So, my advice is to take cell-based metrics of "isomorphism"
> with a grain of salt.
>
> It has already been pointed out that a pure scaling cell deformation
> (one that preserves all the fractional coordinates of all the atoms) does not
> change the structure factors. I would call such a pair of crystals isomorphous.
>
> The origin of the cell-based rule of thumb quoted in Drenth is indeed
> the 1956 paper by Crick and Magdoff that John Cooper shared. But I must
> stress: their calculation, while groundbreaking, was incredibly simplistic. It was
> equivalent to changing the header of a PDB file to a different unit cell, leaving
> all the atoms at the same orthogonal x,y,z positions without regard for crystal
> packing and non-bond clashes. The non-physical-ness of this approach is
> perhaps why noone has ever re-visited it. It is also maximally pessimistic, as
> real crystals are no doubt somewhere in between the harshly rigid
> approximation of Crick & Magdoff and the perfectly soft elasticity that yields no
> change in structure factors at all.
>
> To be fair, I suspect the computer used to do these calculations was
> named Beatrice Magdoff. That is, in 1956 a "computer" was a job description,
> not a device. Magdoff did some amazing things in her career, and this one was
> no doubt a lot of work. I don't blame her and Crick for trying to keep it simple. I
> would have done the same. I also suspect Magdoff would agree that computers
> in 2024 are a bit more powerful than the fastest computers of 1956.
>
> I expect in the coming year that barriers like non-isomorphism will start
> to be overcome. No doubt borrowing from our cryo-EM friends who have been
> stretching, pulling and sharpening 3D images for decades.
>
> Happy New Year everyone!
>
> -James Holton
> MAD Scientist
>
>
> On 12/21/2023 11:37 AM, Tom Peat wrote:
>
>
> Hello All,
>
> I think Randy makes a very good point here- it depends on
> what you are trying to do with your data sets.
> If you are trying to merge them, 'isomorphous' is important for
> this to work. If you are using them for cross crystal averaging, being less
> isomorphous is better (more signal).
>
> James Holton has a story of Louise Johnson collecting data on
> lysozyme (back in the 60's?) where she looked at one specific reflection to
> determine whether the data sets she was collecting would be isomorphous and
> scale. It turns out that although the cell was very similar, the dehydration state
> of the crystal was very important for two lysozyme data sets to scale together.
> The Rmerge for the two dehydration states was something crazy large, like 44%,
> even though under the standard 'rules' (more rules of thumb), one would have
> believed that these data sets should have been 'isomorphous'. For the data sets
> that had the same dehydration state, the data merged with 'typical' statistics of
> lysozyme (like 3-4%).
>
> James will have the details that I do not.
> cheers, tom
> ________________________________
>
> From: CCP4 bulletin board <[log in to unmask]>
> <mailto:[log in to unmask]> on behalf of Randy John Read
> <[log in to unmask]> <mailto:[log in to unmask]>
> Sent: Thursday, December 21, 2023 10:53 PM
> To: [log in to unmask]
> <mailto:[log in to unmask]> <[log in to unmask]>
> <mailto:[log in to unmask]>
> Subject: Re: [ccp4bb] what is isomorphous?
>
>
> [You don't often get email from [log in to unmask]
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>
> I think we’ve strayed a bit from Doeke’s original question
> involving crystals A, B and C, where I think the consensus opinion would be that
> we would refer to crystal C as not being isomorphous to either A or B.
>
> On the question of what “isomorphous” means in the context
> of related crystals, I’m not sure we have complete consensus. I would tend to
> say that any two crystals are isomorphous if they have related unit cells and
> similar fractional coordinates of the atoms, so that (operationally) their
> diffraction patterns are correlated. However, there might be differences of
> opinion on whether two crystals can be considered isomorphous if one has
> exact crystallographic symmetry and the other has pseudosymmetry. (I would
> probably be on the more permissive side here.)
>
> In principle, I suppose being isomorphous (“same shape”)
> should be a binary decision, but in practice we’re interested in the implications
> of the degree to which perfect isomorphism is violated. So I would tend to use
> the term “poorly isomorphous” for a pair where the correlation between the
> diffraction patterns drops off well before the resolution limit. Crick was focused
> on percentage change in cell dimensions, but Bernhard is right that what
> matters is the ratio between the difference in cell lengths and the resolution of
> the data. It’s a bit counter-intuitive, but the effect of the difference between cell
> edges of 20 and 25 is the same as for cell edges of 200 and 205! By the way, the
> first time I learned this was from K. Cowtan and I hadn’t realised it’s also in Jan
> Drenth’s book.
>
> For isomorphous replacement (something some of us dimly
> remember from the days before AlphaFold), being poorly isomorphous is bad,
> but for cross-crystal averaging the more poorly isomorphous the better, because
> the molecular transform is being sampled in different places in reciprocal space.
>
> Best wishes,
>
> Randy Read
>
> > On 21 Dec 2023, at 10:53, Jon Cooper <0000488a26d62010-
> [log in to unmask]> <mailto:0000488a26d62010-dmarc-
> [log in to unmask]> wrote:
> >
> > Hello Harry,
> >
> > I think this is the paper you mean:
> > https://scripts.iucr.org/cgi-bin/paper?S0365110X56002552
> >
> > They gave depressingly low estimates of how much the cell
> dimensions could change in order for isomorphous replacement to still work. In
> reality, unit cells can shrink and swell, but the fractional atomic coordinates
> remain relatively unchanged (right?) so bigger unit cell differences still allow
> the method to work.
> >
> > Best wishes, Jon Cooper. [log in to unmask]
> <mailto:[log in to unmask]>
> >
> > Sent from Proton Mail mobile
> >
> >
> >
> > -------- Original Message --------
> > On 21 Dec 2023, 09:07, Harry Powell < 0000193323b1e616-
> [log in to unmask] <mailto:0000193323b1e616-dmarc-
> [log in to unmask]> > wrote:
> > Hi Didn’t Francis Crick have something to say about this in the
> early 1950s? I’m sure it was published but off the top of my mind I can’t think
> where (one of the more “established” members of this community will be able
> to give chapter and verse)! If you want to read something a little more detailed
> than people have mentioned here, there’s a “Methods in Enzymology” chapter
> by Charlie Carter (?) et al from the early part of this century on the subject -
> again, I can’t remember exactly who or when. Have a good break (which
> reminds me to register for the CCP4 Study Weekend)! Harry > On 21 Dec 2023,
> at 08:04, Tim Gruene wrote: > > Hi Doeke, > > you can take the coordinates of B
> and do a rigid body refinement > against the data from A. If this map is
> sufficient to reproduce model A > (including model building and more
> refinement cycles), then B is > isomorphous to A. You can do this the other way
> round, and the result > may not be the same - hence, the mathematical
> definition of isomorphous > is not identical to the practical use of 'isomorphous'
> structures when > it comes to phasing. You can repeat this for each side of the
> triangle > (each in two directions) in order to label the semantic triangle. > >
> Merry Christmas, more peace on earth and sanity for the elections in > 2024! >
> > Tim > > On Wed, 20 Dec 2023 20:15:17 +0000 "Hekstra, Doeke Romke" >
> wrote: > >> Dear colleagues, >> >> Something to muse over during the holidays:
> >> >> Let's say we have three crystal forms of the same protein, for >> example
> crystallized with different ligands. Crystal forms A and B >> have the same
> crystal packing, except that one unit cell dimension >> differs by, for example,
> 3%. Crystal form C has a different crystal >> packing arrangement altogether.
> What is the right nomenclature to >> describe the relationship between these
> crystal forms? >> >> If A and B are sufficiently different that their phases are >>
> essentially uncorrelated, what do we call them? Near-isomorphous? >> Non-
> isomorphous? Do we need a different term to distinguish them from >> C or do
> we call all three datasets non-isomorphous? >> >> Thanks for helping us resolve
> our semantic tangle. >> >> Happy holidays! >> Doeke >> >> ===== >> >> Doeke
> Hekstra >> Assistant Professor of Molecular & Cellular Biology, and of Applied
> >> Physics (SEAS), Director of Undergraduate Studies, Chemical and >> Physical
> Biology Center for Systems Biology, Harvard University >> 52 Oxford Street,
> NW311 >> Cambridge, MA 02138 >> Office: 617-496-4740 >> Admin: 617-495-
> 5651 (Lin Song) >> >> >> >>
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> -----
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical Research Tel: +44 1223
> 336500
> The Keith Peters Building
> Hills Road E-mail:
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