[buster-discuss] Refining hydrogen atoms

Thu Mar 18 20:44:49 CET 2021

one thing I forgot to mention: aB_hydrogenate -full -ecloud and hydrogenate -full -ecloud wrote identical coordinates of the heme, no matter if I used -l or not.

Wei-Chun

> On 18. Mar 2021, at 16:55, Wei-Chun Kao <wei-chun.kao at biochemie.uni-freiburg.de> wrote:
> 
> Dear Clemens,
> 
> many thanks for your detailed replies, I managed to run buster with -M Ecloud for a project which does not contain prosthetic groups, but I have encountered additional issues with heme while using hydrogenate.
> 
> When I removed all the hydrogen atoms in the heme (HEM), and added them back using hydrogenate (for the sake of using -full -ecloud), I could not refine it in BUSTER with the default restraints since the names of hydrogen atoms did not match. For instance, hydrogenate added HMA1, HMA2, HMA3 to atom CMA, but the HEM.cif in the default BUSTER path (tnt/data/common-compounds and (tnt/data/common-compounds/ecloud) expected HMA, HMAA, and HMAB on CMA. Why hydrogenate added hydrogens to HEM using another atom naming system?
> 
> Even if hydrogenate calls reduce from the molprobity distributed by ccp4, the HEM.cif in the ccp4 monomer library also uses the HMA, HMAA, and HMAB definition as well. Interestingly the HMA1, HMA2 and HMA3 convention is used in the HEM.cif in phenix, and HEC.cif in BUSTER, ccp4 and phenix. 
> 
> By the way, the HEM.cif as well as HEC.cif designate the pyrrole ring A and C as aromatic in BUSTER (see attached picture). The bond lengths appear to follow the resonance structure of porphyrin similar to what was discussed in this ccp4bb thread,
> 
> https://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg36393.html <https://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg36393.html>
> 
> but what is the reason to have two aromatic rings in the restraints?
> 
> Cheers,
> 
> Wei-Chun
> 
> 
> <HEM_Buster.png>
> 
>> On 18. Mar 2021, at 11:04, Clemens Vonrhein <vonrhein at globalphasing.com <mailto:vonrhein at globalphasing.com>> wrote:
>> 
>> Dear Wei-Chun,
>> 
>> On Mon, Mar 15, 2021 at 09:22:14AM +0100, Wei-Chun Kao wrote:
>>> I am refining a few similar structures ranging from 1.0 to 2.5 Å
>>> resolution. I added hydrogen atoms to the models and after
>>> refinement, the occupancies of all hydrogen atoms were set to
>>> zero. It is clear from refine.log that “All hydrogens are given with
>>> zero occupancy, i.e. invisible to X-Ray term”.
>> 
>> Correct - so they only contribute to the geometry term (at the nuclear
>> position by default).
>> 
>>> I also repeated the 3a4r example in a BUSTER tutorial
>>> 
>>> https://www.globalphasing.com/buster/wiki/index.cgi?AutobusterHydrogens <https://www.globalphasing.com/buster/wiki/index.cgi?AutobusterHydrogens>
>>> 
>>> with exactly the same commands, except that I needed to use the
>>> -full option of “hydrogenate" which is not stated (or was not
>>> available then) to keep the occupancies of the existing hydrogen
>>> atoms in 3a4r.pdb.
>> 
>> Ah yes - thanks for highlighting the need to update those tutorial
>> pages asap!
>> 
>>> After refinement, the occupancies of hydrogen atoms were set to
>>> zero, which is perhaps not supposed to be the case in the tutorial?
>> 
>> It was at the time - since we didn't have support for electron-cloud
>> position of hydrogens (this was added in 2019). If hydrogens are
>> placed at the nuclear position it doesn't make sense to have them
>> visible to the X-Ray term (i.e. with occupancy > 0): X-Ray see
>> hydrogen electrons slightly shifted along the covalent bond towards
>> the parent atom (see [1] and [2]).
>> 
>> However, with an electron-cloud model the situation changes: the
>> hydrogens are in a position adequate for X-Ray data and can be used at
>> full occupancy (i.e. same occupancy as parent atom). You can decide on
>> which model to use e.g. via
>> 
>>  aB_hydrogenate -p your.pdb               -o your_nucl.pdb
>>  refine -p your_nucl.pdb ...
>> 
>>  aB_hydrogenate -p your.pdb -full -ecloud -o your_ecld.pdb
>>  refine -p your_ecld.pdb -M Ecloud ...
>> 
>>> I got “best refinement in BUSTER reached for FP,SIGFP with R/Rfree
>>> 0.2178/0.2291, which are higher than the results shown in the
>>> tutorial 0.1982/0.2120. I guess it’s (partly) the effect of
>>> excluding hydrogen atoms for refinement?
>> 
>> Most likely that effect is due to the change of default R-value we
>> report since the December 2020 version (R-values now based on F(calc)
>> instead of F(expect) as before - see [3]). We definitely need to
>> update that tutorial!
>> 
>>> I have two sets of questions:
>>> 
>>> 1. Is there a way to keep the occupancies of hydrogen atoms to 1.0
>>>   for my 1.0-Å resolution dataset?
>> 
>> Absolutely, by using the "-M Ecloud" macro in "refine" (see "refine -M
>> show" to see the various parameter this macro sets). If you have a PDB
>> file with hydrogens at full occupancy already you are ready to go. If
>> you have no hydrogens in your model PDB file yet, use "aB_hydrogenate"
>> with the "-full" and "-ecloud" switch. Please note that
>> "aB_hydrogenate" calls the original "hydrogenate" tool (which in turn
>> calls Reduce from Molprobity suite as distributed by CCP4), but adds a
>> few fix-up functions as well as handling non-standard residues more
>> automatically.
>> 
>>>   Interestingly the occupancies of all hydrogen atoms in 3a4r.pdb
>>>   are all 0.88.
>> 
>> Have we mentioned the need for updating that tutorial page? :-)
>> 
>> You could run
>> 
>>  sed "s/  0\.88\(.* H[ ]*\)$/  1.00\1/g" 3a4r.pdb > 3a4r_occ1.pdb
>> 
>> to change all occ=0.88 hydrogens to occ=1.
>> 
>>>   Is it even sensible to refine the occupancies of hydrogen atoms?
>> 
>> No - but their occupancies needs to stay in sycn with the parent atom
>> (if this is in alternate conformations and/or has its occupancy
>> refined).
>> 
>>> 2. For the 2.0-ish Ā resolution datasets, perhaps it is a better
>>> idea to set the occupancies of hydrogen atoms to zero?
>> 
>> It depends a bit on the data quality (one 2A project is not always the
>> same as another 2A structure). You could definitely try with full
>> ("-ecloud -full" in aB_hydrogenate and "-M ecloud" in refine)
>> occupancy or with occ=0. When getting closer to ~1.5A the benefits of
>> using the e-cloud/full model usually become more pronounced.
>> 
>>> Is this the way BUSTER treats hydrogen atoms as riding model?
>> 
>> Buster doesn't really use a so-called "riding model" (as in: hydrogens
>> contribute to geometry/x-ray function values but their positions are
>> not refined themselves but calculated based on the parent atom). All
>> hydrogens will be refined like any other atom: either through geoemtry
>> terms alone (nuclear position and occ=0) or against geometry and X-Ray
>> together (e-cloud and full occupancy).
>> 
>> One thing to remember after a BUSTER refinement including hydrogens:
>> during model validation you might need to tell programs to keep the
>> hydrogens as they are and to use the correct (ideal) hydrogen
>> position. E.g.
>> 
>>  molprobity.molprobity keep_hydrogens=True nuclear=False refine.pdb
>> 
>> if you used the full occupancy, e-cloud model.
>> 
>>> Will the
>>> zero-occupancy hydrogens still improve the geometry of the model at
>>> this resolution?
>> 
>> Yes: they are fully visible to the geometry term and therefore also to
>> the contact term - resulting in better placement of e.g. side-chain
>> rotamers etc.
>> 
>>> The BUSTER version is snapshot_20210224, the operating system is CentOS 7.
>> 
>> Good.
>> 
>> If there are further questions regarding BUSTER and hydrogens, please
>> let us know!
>> 
>> Cheers
>> 
>> Clemens
>> 
>> [1] https://onlinelibrary.wiley.com/doi/pdf/10.1002/pro.3330 <https://onlinelibrary.wiley.com/doi/pdf/10.1002/pro.3330>
>> [2] https://journals.iucr.org/d/issues/2018/08/00/mn5114/mn5114fig8.html <https://journals.iucr.org/d/issues/2018/08/00/mn5114/mn5114fig8.html>
>> [3] https://www.globalphasing.com/buster/wiki/index.cgi?BusterRvalues <https://www.globalphasing.com/buster/wiki/index.cgi?BusterRvalues>
>> 
>> -- 
>> 
>> *--------------------------------------------------------------
>> * Clemens Vonrhein, Ph.D.     vonrhein AT GlobalPhasing DOT com
>> * Global Phasing Ltd., Sheraton House, Castle Park 
>> * Cambridge CB3 0AX, UK                   www.globalphasing.com <http://www.globalphasing.com/>
>> *--------------------------------------------------------------
> 
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