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Paul Adams
We’ve started the recording.

chmjm
Thank you

Nic Steussy
resolution recommendations for anisotropic or tls refinement?

LC
for high anisotropic data try Staraniso website and check if anisotropic map are better than isotropic map

LC
for me tls is just a way of decrease R factor for things you have not yet modelled, so bad

Monika Bjelcic
is it possible to run xtriage on serial data?

Misbha
how does the meaning of the occupancy differ in case of ligand. For non-covalent ligands what should be the occupancy fixed at at the beginning of the refinement.

student
At which point is better to run TLS parameter?

student
Is there any way if I have two molecules of ligand in my binding pocket but not appear as the same occupancy so is phenix can tell us or optimize the occupancy of the two copy of ligand

Nigel W. Moriarty
Occupancy for covalent ligands can be set to something like 20 or similar to the bonded protein atom. It may be that the protein is too high so starting at 20 or 30 is fine

Vatsal Purohit
How would you approach refining a structure with multiple ligand populations at the same position (i.e. multiple intermediates in the same crystal with different occupancy)?

Almudena Ponce Salvatierra
If the data are very anisotropic (i.e. 2.5 Angstroms, 4 and 4 along x, y z axis), but have been elliptically truncated, is there anything especial that one needs to think of when refining? Model is close to final.

Nigel W. Moriarty
you can refine two ligand occupancies

Nigel W. Moriarty
read this http://phenix-online.org/newsletter/CCN_2015_07.pdf#page=12

Anil Sohail
There are two types of options for simulated annealing (Cartesian and Torsion angles) what is the difference and when to use ?

LT Zhai
To

Nigel W. Moriarty
Two different ligands in the pocket is similar to example 5

Nigel W. Moriarty
Of course, the accuracy of the answer depends on the resolution

Nigel W. Moriarty
bioarx https://www.biorxiv.org/content/10.1101/2020.03.26.010587v1

Nigel W. Moriarty
journal https://www.cell.com/structure/fulltext/S0969-2126(20)30287-2

Scott Classen
Great talk Pavel!

Nic Steussy
thanks very useful

LC
Hi Pavel, Tanks for this very nice talk (basic teaching, but very nice), as usual. I’d like to ask about anisotropy and how phenix.refine deals with it. Apart my specific question, I think that anisotropy is not well understood by most people, so general & specific comments should be good for all. Anisotropic scaled dataset (from Staraniso) can have anisotropic cut-off reflections marked as non measured or not be present in the mtz (as far as I remember) . How phenix.refine deals with those reflections during the refinement: filled / unfilled? Any difference between Staraniso anisotropic cut-off data and normal spherical cut-off data? (I know that phenix.refine output filled/unfilled map) Thanks, Lionel

Agni Ghosh
For a 0.9 A dataset, can I use Phenix or do I need to use packages like Shelx?

Anil Sohail
Thank you for the wonderful talk.

Nigel W. Moriarty
newsletter http://phenix-online.org/newsletter/

Nigel W. Moriarty
Phenix will work well for 0.9Å data

Jan Gebauer
Is it possible to subscribe to the newsletter? (couldn't find a way...) Or get some way of reminder when a new newsletter comes out?

Claudia Lucía Millán Nebot
it is announced in the phenix jan

Nigel W. Moriarty
I announce it on the phenixBB

Agni Ghosh
Thank you, Nigel.

Claudia Lucía Millán Nebot
Yes Paul point is very relevant. To use in phasing please do use original data, not truncated

Nigel W. Moriarty
There are special features for hi-res refinements that you should become aware of

LC
Hi Pavel, Tanks for this very nice talk (basic teaching, but very nice), as usual. I’d like to ask about anisotropy and how phenix.refine deals with it. Apart my specific question, I think that anisotropy is not well understood by most people, so general & specific comments should be good for all. Anisotropic scaled dataset (from Staraniso) can have anisotropic cut-off reflections marked as non measured or not be present in the mtz (as far as I remember) . How phenix.refine deals with those reflections during the refinement: filled / unfilled? Any difference between Staraniso anisotropic cut-off data and normal spherical cut-off data? (I know that phenix.refine output filled/unfilled map) Thanks, Lionel

Almudena Ponce Salvatierra
Super! Thank you very much! Understood!

Dorothee Liebschner
For high resolution refinement, the documentation has some useful info:

Dorothee Liebschner
http://cci.lbl.gov/ceres/goto_entry/7a6b_11669/10_2020

Dorothee Liebschner
wrong link

Dorothee Liebschner
https://www.phenix-online.org/documentation/reference/refinement.html#refinement-at-high-resolution-higher-than-approx-1-0-angstrom

Agni Ghosh
Thank you, Dorothee!

Andrea Pica
Can I make Phenix refinement run faster?

vserrano
I have question about merge data sets in phenix,, how you decide what data can be merged, and hoe is the scaling of data sets done?

Maria Rutkiewicz
When it comes to high resolution data (0.84A) I have realized that the B factors are not correctly refined and as a result a lot of positive peaks (green map) are observed for instance on main chain atoms. The data is very high accuracy and I/sigI in the highest resolution shell about 5 (the detector couldn't have been moved closer) How can we solve this issue?

Claudia Lucía Millán Nebot
Mmm I’m also interested in the question about merging of datasets in phenix! :)

Mark Bostock
How do you generate Rfree flags if not already present in the mtz file?

Dorothee Liebschner
@Maria: It may be that the B-factors are in a wrong minimum. You could reset them (f.ex. use phenix.pdbtools) and refine again. Also, are you using isotropic or anisotropic B-factors?

Dorothee Liebschner
@Mark:

Dorothee Liebschner
You can generate Rfree flags

Dorothee Liebschner
https://www.phenix-online.org/documentation/reference/reflection_file_editor.html#dealing-with-r-free-flags

Alex Chu
Is it appropriate to truncate data to try for a lower Rfactor/Rfree? If not, what are some strategies for getting lower Rfactor/Rfree?

Nigel W. Moriarty
@Mark phenix.refine will also do it if you set the option

Mark Bostock
@Dorothee, @Nigel - thanks!

Dorothee Liebschner
@Alex: it is not advisable to truncate data for the sole purpose of lowering Rwork/Rfree. Truncating should be only done if the data don’t have enough information content. Instead, I suggest 1) investigate if the data have some underlying issues (twinning, radiation damage, etc) 2) think about the refinement strategy (what parameters are refined) 3) look at the maps: are there unmodeled regions or are the maps “clean”?

Dorothee Liebschner
@Alex (continued): You should also look at model quality and model-vs-data fit. R-factors are not the sole measures for refinement outcome.

Alex Chu
@Dorothee - thank you!

Jacob W
On the topic of NCS and whether we should apply it - is there a percentage of residues on each chain that we should reach to determine whether or not we apply NCS restraints?

Nic Steussy
coment on optimize xray?

Rajnandani Kashyap
i have a dataset where i see 2 molecules in the asymmetric unit but they are distant apart? I tried solving the data in different space group to see of its correct but data fots very well in this space group..Appreciate for any suggestions

Rajnandani Kashyap
fits*

Pavel Afonine
“Is it appropriate to truncate data to try for a lower Rfactor/Rfree? If not, what are some strategies for getting lower Rfactor/Rfree?” — No, bad idea.

vserrano
How can one refine one particular bond lengh in a ligand, during refinement run?

Pavel Afonine
“resolution recommendations for anisotropic or tls refinement?” — Indivisual anisotropic: 1.7A or better, TLS otherwise.

Pavel Afonine
@Lionel, re STARANISO: refine with whatever let’s you to progress as far as you can, but switch to original data towards the end.

Pavel Afonine
“At which point is better to run TLS parameter?”: towards the end of refinement. Or definitely not in the begining!

Paul Adams
“resolution recommendations for anisotropic or tls refinement?” - remember that individual anisotropic add 5 more parameters per atom, so a lot of data is needed. Consider that you’d like 3 or more reflections per parameter in refinement, so with N atoms that would be 3x9xN reflections (approximately) for anisotropic displacements and coordinate refinement.

Pavel Afonine
“On the topic of NCS and whether we should apply it - is there a percentage of residues on each chain that we should reach to determine whether or not we apply NCS restraints?” — No black/white answer here. Try both and see what works best.

Maria Rutkiewicz
@Dorothee Liebschner: It is refined anisotropically but I also tried isotropically. I have reseted B-factors to some values (like3, 6 etc) but at the end the map shows positive peaks on many atoms.

Paul Adams
On the topic of NCS and whether we should apply it - is there a percentage of residues on each chain that we should reach to determine whether or not we apply NCS restraints?” - adding any restraints from NCS should help, but the fewer the restraints the less impact on refinement. The default cutoff is 85% sequence identity. If you have chains that are more different then you might need to change the cutoff.

Pavel Afonine
“how does the meaning of the occupancy differ in case of ligand. For non-covalent ligands what should be the occupancy fixed at at the beginning of the refinement.” — occupancy reflects the probability of the ligand to be there, which ranges from 0 to 1. It is likely the ligand is partially occupied in which case occupancy should be refined.

Pavel Afonine
“How do you generate Rfree flags if not already present in the mtz file?” — you should do it first thing once you got the data. Reflection file editor in Phenix GUI is perhaps the best place to do it!

Dorothee Liebschner
@Maria: I see. If you want, I can have a look at your data. If you can share, you can send them to dcliebschner@lbl.gov. I would need all relevant files to do refinement: model, data, ligand restraints, non-default options you used

Pavel Afonine
“How can one refine one particular bond lengh in a ligand, during refinement run?” — Not sure what this means. Try to come back with this questions again at Q&A session!

Pavel Afonine
“Is there any way if I have two molecules of ligand in my binding pocket but not appear as the same occupancy so is phenix can tell us or optimize the occupancy of the two copy of ligand” — Good question. Yes, it is possible, this topic is covered here: http://phenix-online.org/newsletter/CCN_2015_07.pdf#page=12

Andrea Pica
Links to residues are applied automatically... at which level? How to generate and modify the distance, angles, dihedrals between protein and ligands in general? Also, in general, do we need a cif file for the ligands and one cif to specify the connectivity with the particular protein residue it is bound to?

Pavel Afonine
“For a 0.9 A dataset, can I use Phenix or do I need to use packages like Shelx?
” — to add on this.. This may need some parameter tweaking. So please reach us out if you need any help and we will make sure you have best parameter setup for refinement.

Andrea Flores
Is it always recommended to use at some point Amber restrains or are there more particular cases better suited for it?

Pavel Afonine
“Links to residues are applied automatically... at which level? How to generate and modify the distance, angles, dihedrals between protein and ligands in general? Also, in general, do we need a cif file for the ligands and one cif to specify the connectivity with the particular protein residue it is bound to?” — automatic linking totally relies on input model geometry. If atoms are close enough and it makes chemical sense, then the program will try to link them. If not, it will leave them alone. In rare instances it may link what’s not supposed to be linked. So better check. *geo file out of refinement lists all the links and geometry restraints so it’s the best place to check!

Andrea Pica
Thanks Pavel. What if the atoms to be linked are far and I want to force the link between them. How do I control the geomentry of the bond?

Pavel Afonine
“Thanks Pavel. What if the atoms to be linked are far and I want to force the link between them. How do I control the geomentry of the bond?” — yes, you can do it by defining custom bonds in refinement: http://phenix-online.org/documentation/reference/refinement.html#definition-of-custom-bonds-and-angles

vserrano
If ou make a modification in .geo file, rather then cif file, hoe do you include the .geo file in the next refinement cycle using gui version?

Dorothee Liebschner
@vserrano: the .geo file is only informative output. It cannot be used to change restraints. You will have to define custom bonds in the edits file.

Rajnandani Kashyap
i have a dataset where i see 2 molecules in the asymmetric unit but they are distant apart? I tried solving the data in different space group to see of its correct but data fits very well in this space group, lower Rfactor/Rfree..Appreciate for any suggestions

vserrano
Thanks.

Paul Adams
“i have a dataset where i see 2 molecules in the asymmetric unit but they are distant apart?” - one of the molecules probably needs to have a symmetry operation applied to put it close to the other. I think you can do this in Coot.

Rajnandani Kashyap
@Paul Adams thanks ..any option you can elaborate on

Najeeb Ullah
protein molecule with 12 chains, each chain has ribose 5-phosphate bound, but density of the ligand (R5P) is different in each chain, mean ligand can be ribose and/or can be ribofuranose, how one can go with eLBOW and refinement?

Pavel Afonine
“Can I make Phenix refinement run faster?” — In general it does a lot of things to save you from doing them yourself manually. Do you have an example where runtime overhead is unreasonable vs outcome?

Andrea Pica
What is your recommendation to generate bond information to create a data_link cif file between a compound and a protein residue?

Vatsal Purohit
Can you point us to the most updated list of ligand restraints available on the latest version of phenix?

Pavel Afonine
“Can you point us to the most updated list of ligand restraints available on the latest version of phenix?” — Look in your Phenix sources in phenix/modules/chem_data

Avradip
at what stage of refinement after molecular replacement should one start looking for ligand density

student
Which file should be provided as you mentioned for the two same ligand in the pocket but different occupancy?

Pavel Afonine
“at what stage of refinement after molecular replacement should one start looking for ligand density” — Keep eye on the pocket constantly from start to end. But model the ligand towards the very end when all else is as good as possible.

Najeeb Ullah
and the ligand (R5P) binds through anomeric carbon

Avradip
Thanks

vserrano
Thanks Nigel, great talk

Andrea Pica
Thanks Nigel. Pavel also replied in the chat. I will get in touch if I have troubles.

student
How can we fix the occupancy of two same ligand in the same bonding pocket?

Rajnandani Kashyap
How we be 100% sure that the ligand density we are looking for is the exact molecule that you think it is..in my case I doubt the ligand density with cryosolution ingredient..i checked the crystals mass spec after washing with the mother liquor but it gives mass of the ligand..is it acceptable?

Andrea Flores
Thank you!

Najeeb Ullah
@Nigel it is R5P with different confirmations, density is not clear for Ribose and ribofuranose I will connect via email for details

Giang Nguyen
Can you comment on Polder maps for ligands?

Hengjun Cui
What is the best way to prove the existence of a ligand in the structure?

student
Yes you right we can fix the occupancy in coot but we run in phenix its change the occupancy half and half again

Dorothee Liebschner
@Giang: https://www.phenix-online.org/documentation/reference/polder.html

Pavel Afonine
“What is the best way to prove the existence of a ligand in the structure?” — Use Polder OMIT map and its interpretation.

Hengjun Cui
How about the bias from the model provided then? With simulated annealing in addition?

Pavel Afonine
“Yes you right we can fix the occupancy in coot but we run in phenix its change the occupancy half and half again” — phenix.refine is supposed to refine the occupancy. So if it refines it to 50/50 then perhaps this is what it is!

Misbha
what to do in case where a part of the ligand doesn’t have density but the rest has very good density and fits well?

Jiang Yin
for ligand ID tool, where to find in phenix?

Pavel Afonine
“How about the bias from the model provided then? With simulated annealing in addition?” — If you have not built the ligand in the first place then there is no bias.

Rajnandani Kashyap
thank you

student
Thanks everyone

Giang Nguyen
Thanks!

Silvia Napolitano
Thanks Nigel for your talk! You also answered my initial question about SS bonds (it worked now :) )

Jiang Yin
also very interested in tools that helps with interpreting extraneous densities connected to protein residues, i.e., possible covalent modifications?

Nigel W. Moriarty
If you have extraneous densities, it could be covalent mods but we don’t have an easy solution

Nigel W. Moriarty
I have some ideas so I’d be happy to take a look

Nigel W. Moriarty
@Silvia Glad it worked

lwhung
Ligand identification tool is under the “ligands” tab.

Nigel W. Moriarty
@Najeeb I can help with your two ligand situation

Nika Žibrat
I am solving a structure where a ligand is a disaccharide. Previously solved structure of the same protein demonstrated the shift in certain regions upon binding of a monosaccharide. My protein exhibits the exact conformational shift expected for the binding of the ligand, however the electron density is not there in sufficient area to fit the disaccharide. The area of green density only roughly corresponds to a monosaccharide moiety. MS experiments confirmed the disaccharide is stil intact. Resolution is 2. The ligand was soaked, not co-crystallized. Any suggestions?

jossaf
I have a structure at 1.3A resolutions and most of it looks good and I am at the end of model refinement but I the clashscore is high.

jossaf
is there any easy why to deal with clashscores?

Dorothee Liebschner
@Jossaf: Did you look at the clashes in Coot and did you try to fix them manually?

jossaf
yes and some of them does not look like clashes because they have good defined electron density maps

Nigel W. Moriarty
@Nika Fit the monosaccharide and refine. The density may appear. Also do a Polder map of the monosaccharide to see if you can see the second

Nigel W. Moriarty
@Nika you can also then fit the second and do a Polder of the second and so what happens

Nika Žibrat
Thanks!

jossaf
and most of the time sidechain and water molecule should be where they are

Rajnandani Kashyap
Please educate us about the ethics of publishing structures looking at the ascertainity around the ligand density..I often have seen criticism over published structure with false ligand density..as a newbie I really want to hear the experienced

Vincent Chen
@jossaf are there other errors (rama outliers, rotamer outliers, etc) near where there are clashes? sometimes clusters of errors can help identify areas that have a problem that needs to be fixed

Dorothee Liebschner
@Jossaf: Then fix first those where the density is not so good. The Phenix GUI orders the clashes according to overlap. This allows finding the worst overlaps.

Nigel W. Moriarty
@Rajnandani Run Polder to see what it says. Read the Twilight paper to see what Rupp says should be there.

Dorothee Liebschner
@Rjnandani: 10.1107/S0907444912044423 and follow up papers

Rajnandani Kashyap
thanks

Nigel W. Moriarty
@Rajnandani BTW, we are working on a suite of tests so we would like to see your example if you’d like to share

jossaf
@vincent in some parts yes, but in some no

jossaf
@dorothee thank I will try to re-look them

Rajnandani Kashyap
@Nigel W. Moriarty Sure

Dorothee Liebschner
@Rajnandani: yes, examples for unclear ligands would be helpful

Gye Won Han
Can you explain 'RSRZ' outliers in PDB validation?

jossaf
I have a question about a amount of water in a structure, I have 1.2A resolution, my protein has 400 AA and is highly soluble in sodium buffers at pH 5. What is a good amount of water? I am adding water manually with COOT after phenix.refine and I have 340 H2O molecules is this enough? I am looking the electron density map at 1.40 rmsd.

Gye Won Han
Phenix can incorporate it? gyewon.han@usc.edu

Gye Won Han
PDB validation reports always list RSRZ>2.

Paul Emsley
rule of thumb: 2 waters/residue

Pavel Afonine
“Can you explain 'RSRZ' outliers in PDB validation?” — This is a flawed metric as it is implemented in PDB. Tom and others have reported issues with this metric and never heard back from PDB. So.. personally I think as long as usual CC is good you are good.

Gye Won Han
I totally agree with you regarding RSRZ outliers.

jossaf
@paul thank you very much

Nic Steussy
clap clap

Laura Pacoste
Thanks Christopher, very nice talk!

Rajnandani Kashyap
👏

Maria Hrmova
Thank you Christopher, awesome talk!

Nic Steussy
could you comment on adjusting the wxc or wxc_scale factors to improved overall structural bonds and angles.

Paul Emsley
Where do you stand on the replacement of the refined hydrogen atoms when determining the clashscore?

jossaf
true I have a problem in one loop

jossaf
my b factors are around 100

jossaf
thank you a lot

Gihan Ketawala
When are the talks gonna be online?

Nigel W. Moriarty
@Gihan In a few days

Shymaa
Question about refinement: I have twinned data and I did first refinement cycle using the twin law in Phenix. Would it be logical to use the refined reflection data from the first cycle in the second cycle of refinement and not use the twin option again in phenix? Can someone comment how the twinned data should be refined.

Rajnandani Kashyap
comment on Temperature factor variance for validation of structure

Paul Emsley
(hehe)

Dorothee Liebschner
Hopefully, I can edit and upload the videos until next week.

Lucia Dello Iacono
Do you plan to implement a specific software for sugar conformation validation in Phenix similar to Privateer?

Vatsal Purohit
Can you comment a bit on the autobuild refinement feature? I am currently attempting to use it to rebuild flexible regions of my protein that don't always agree with my MR search model. In autobuild, what is the difference between the experimental map and initial map? Which map is best for rebuilding flexible regions that are too big or tricky to fix by hand on coot? Lastly, how do you preserve the numbering on your initial model when using autobuild? - I find that my model gets renumbered after the program works through the flexible regions that might have scantier density.

Eta Isiorho
Is there anything different you need to do for the ‘final’ refinement when you

Jiang Yin
A particular problem that I recently encountered is how to resolve strong residual peaks (>5 sig) in the difference map after fitting a disulfide bridge between two cysteines. The geometry for the SS is okay to the eye and well fitted in density but there are still residual densities in the difference map. I just heard from someone that many times Zn2+ could be mistaken as -SS-. I am asking the experts here how often do you encounter this, if at all?

Eta Isiorho
Re ready to upload

Nigel W. Moriarty
@Lucia Yes

Eta Isiorho
Sorry, let me type my question again, is there anything different you need to do for the ‘final’ refinement when you’re ready to deposit?

Dorothee Liebschner
@Jiang: It could be radiation damage as well

Nigel W. Moriarty
@Eta To deposit, you should use the mmCIF from refinement in a the PDB model deposition tool to add the sequence and then send the mmCIF to the PDB OneDep

Dorothee Liebschner
@ Jiang: see for example 10.1073/pnas.97.2.623

Nic Steussy
thanks

Eta Isiorho
Thank you

Nigel W. Moriarty
@Eta You should use a very recent version of Phenix to get the smoothest process

Lothar
As completeness of dealing with all density is beneficial to the model that ends up as coordinates in the pdb file, are there thoughts on implementing the Squeeze procedure that exists for small molecules ? I realize that this has the potential to open cans and cans of worms but if done properly it may help. There are lots of structure with large unmodeled positive density in the data base.

jossaf
When we see after the phenix.refine the increase in R factors does it mean that we used inappropriate refine strategy or we are overfitting data ?

jossaf
I am at the end of refinement with R work 0.1554 and R free 0.1839

Vatsal Purohit
1 more question about ligand occupancy - If a region that indicates a bond between ligands shows 2Fo-Fc and red Fo-Fc density can that indicate a partial occupancy of that bond? Can a disappearance of the Fo-Fc density at lower occupancy further indicate that?

Lucia Dello Iacono
@Nigel, thanks for confirming that you will include a software for sugar validation in Phenix suite. As far as I know Privateer does not work with oligosaccharides made only by furanose units (at least for modified sugar which are not previously present in the PDB list of ligands). Will your software be able to overcome this issue? Do you know if another software can help me in the meanwhile?

Paul Emsley
It is a small molecule refinement technique.

Paul Emsley
Unmodelled (non-atomic) density is added back to the "model"

Billy Poon
I found this reference for PLATON SQUEEZE (https://doi.org/10.1107/S2053229614024929)

jossaf
this are starting R free is 0.1898

jossaf
final

Vatsal Purohit
Alright, thanks! Will share the file to phenixBB to request more input.

Maria Hrmova
How do you decide on occupancy, if you refine the same sugar in two conformations (4C1 and 1S3 sugar conformations). Do you experiment with numbers, say 0.0 and 0.5, and then 0.3 and 0.7? How do you decide on the correct ratio?

Lothar
Thanks Paul and Billy, this is what I meant.

jossaf
ok thank you

Nigel W. Moriarty
http://www.glycosciences.de/tools/pdb-care/

Jacqueline Vitali
I typically use coot to make cif files for ligands. In case of Zn—his bonds or Zn—water or Zn—Asp and Zn-Glu does one need to restrain lengths and angles at ~2A res?

Lucia Dello Iacono
Many thanks

Anil Sohail
Many thanks for a wonderful workshop :)

jossaf
Many thanks for a wonderful workshop :)

Lilian González Segura
Thanks!

Silvia Napolitano
Thanks for this useful workshop!

Lothar
Thanks!

Sarah Meinhold
thank you

Christoph Giese
thanks for the excellent workshop! keep it up

Paul Emsley
Thanks you. Good night :-)

Jacqueline Vitali
Many thanks for the session. it was great.

Vatsal Purohit
Thanks a lot for the great workshop!

Jacqueline Vitali
we can save it individually

Maria Hrmova
Thank you for an excellent webinars.