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Dear jared,
Thank you so much for the very detailed reply – very much appreciated!
For some reason, pose_from_saccharide_sequence worked after restarting my IDE, so perhaps I had initialited it wrong the first time.
I have played a bit around with the different methods and movers, and I am almost able to do everything I want to do. I have however run into into a problem: I cannot get sialic acids to work, even though two are listed in ‘codes_to_roots.map’:
# Ketononoses (e.g., Sialic Acids)
Neu neuramin * 2 # D inherent
Kdn keto-deoxy-nonulon * 2 # D inherentSialic acids have nine carbons but only have five carbons in the ring ( https://en.wikipedia.org/wiki/Sialic_acid ). I would therefore expect ”a-Neuf5Ac” to work but is does not. The Error I get is:
ERROR: Unrecognized residue ->4)-alpha-Neuf:5-Ac when building saccharide sequence a-Neuf5Ac-(2->3)Can anyone help?
Kind regards,
Martin
EDIT:
it should be ‘p’ when there are five carbons in the ring, but if I try ”a-Neup5Ac” I also get an error
EDIT 2:
I got it working using ‘->
-a-Neup5Ac-(2->3)’ so it was the default value of ->4)… which caused it not to work. Like I said, I am still a rookie with glycans, so not always super on top of the correct settings for the different glycans 
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I usually run my scripts on our cluster with the command: python <script.py> > pyros.log
I am therefore not sure it has to do with the kernel
Kind regards,
Martin
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Hello again,
I have been able to modifiy our MX structure, albeit with some errors, failures and warnings from GlycanTreeModeler. I would also like to use standard movers like SmallMover and ShearMover to model two hinge regions, which has also proved difficult. I hope it is OK I keep asking the questions in this thread, otherwise I am happy to make a new one.
Regading GlycanTreeModeler:
After adding the glycosylations using SimpleGlycosylateMover, I call GlycanTreeModeler which succesfully minimise the structure, although I have to call it many times (> 20) to get reasonable minimisation and even after 100 iterations are there problematic tryptophan geomtries/bond lengths (Trp can have mannose added).
The warnings I tipycally get are of the sort:
protocols.carbohydrates.GlycanTreeMinMover: Minimizing from carbohydrate root: 53
protocols.carbohydrates.GlycanTreeMinMover: Minimizing 3 residues
core.conformation.Residue: [ WARNING ] There are no adjacent heavy atoms to atom index 13!
core.conformation.Residue: [ WARNING ] There are no adjacent heavy atoms to atom index 1!
core.conformation.Conformation: [ WARNING ] Branch 1does not have enough heavy atoms about its connection to define a torsion angle!
core.conformation.Residue: [ WARNING ] There are no adjacent heavy atoms to atom index 14!
core.conformation.Residue: [ WARNING ] There are no adjacent heavy atoms to atom index 1!
core.conformation.Conformation: [ WARNING ] Branch 2does not have enough heavy atoms about its connection to define a torsion angle!
core.conformation.Residue: [ WARNING ] There are no adjacent heavy atoms to atom index 13!
core.conformation.Residue: [ WARNING ] There are no adjacent heavy atoms to atom index 1!
core.conformation.Conformation: [ WARNING ] Branch 1does not have enough heavy atoms about its connection to define a torsion angle!
core.conformation.Residue: [ WARNING ] There are no adjacent heavy atoms to atom index 14!
core.conformation.Residue: [ WARNING ] There are no adjacent heavy atoms to atom index 1!
core.conformation.Conformation: [ WARNING ] Branch 2does not have enough heavy atoms about its connection to define a torsion angle!
protocols.carbohydrates.LinkageConformerMover: Sampling alpha-L-Fucp-(?->6)-beta-D-GlcpNAc- linkage
protocols.carbohydrates.LinkageConformerMover: Sampling conformer 2 which has a population of 0.1813
protocols.carbohydrates.LinkageConformerMover: Complete
...
core.pack.task: Packer task: initialize from command line()
core.pack.rotamer_set.RotamerSet_: [ WARNING ] including current in order to get at least 1 rotamer !!!!!! 4 ASN:N-glycosylated
core.pack.rotamer_set.RotamerSet_: [ WARNING ] including current in order to get at least 1 rotamer !!!!!! 23 SER:O-conjugated
core.pack.rotamer_set.RotamerSet_: [ WARNING ] including current in order to get at least 1 rotamer !!!!!! 26 SER:O-conjugated
core.pack.rotamer_set.RotamerSet_: [ WARNING ] including current in order to get at least 1 rotamer !!!!!! 42 TRP:C-glycosylated
core.pack.rotamer_set.RotamerSet_: [ WARNING ] including current in order to get at least 1 rotamer !!!!!! 45 TRP:C-glycosylated
core.pack.rotamer_set.RotamerSet_: [ WARNING ] including current in order to get at least 1 rotamer !!!!!! 48 TRP:C-glycosylatedand the failures/errors I get:
protocols.carbohydrates.LinkageConformerMover: Sampling beta-D-Glcp-(?->3)-alpha-L-Fucp- linkage
protocols.carbohydrates.LinkageConformerMover: no conformer found. Using sugar BB sampler if possible.
protocols.carbohydrates.GlycanTreeSampler: Last mover failed. Continueing!
protocols.carbohydrates.LinkageConformerMover: Sampling alpha-D-Manp-(?TRP linkage
protocols.carbohydrates.LinkageConformerMover: no conformer found. Using sugar BB sampler if possible.
protocols.carbohydrates.LinkageConformerMover: Cannot run sugar BB Sampler on linkage as linkage as a protein-glycan linkage. Skipping
protocols.simple_moves.bb_sampler.SugarBBSampler: 81 81 A torsion: 2
protocols.simple_moves.bb_sampler.SugarBBSampler: No data for linkage. Either this is psi and previous residue is not carbohydrate or we do not have a pyranose ring in the previous residue.
protocols.simple_moves.BBDihedralSamplerMover: [ ERROR ] Could not set torsion for resnum 81
protocols.carbohydrates.GlycanTreeSampler: Last mover failed. Continueing!To investigate further, I made a simply dummy pose (a pose_from_sequence) which was a straight line and tried to add the glycosilation to that – perhaps some of the problems stem from the glycosylations being added in regions without so much space around it. Yet, I still ran into problems. I have attached a screenshot of wierd Trp geomtry/bond lengths, with the Trp sitting next to Ala so there should be plenty of space.
being unfamiliar with GlycanTreeModeler, I thought that maybe by using standard relaxtion movers like MinMover I could at least fix the tryptophan geometries (with a movemap with set_chi(True)), which in turn would allow GlycanTreeModeler to fix the errors with the glycans (if any is present, SimpleGlycosylateMover does not produce any warnings). That is, perhaps if I iteratively called GlycanTreeModeler and MinMover then problems would might go away. This proved not to be the case, at least not with 20 iterations – perhaps I could improve them by iterating longer.
Regarding other movers (e.g. SmallMover and ShearMover)
The main problem I have had is that I get zero accepted moves regardless of how high I put kT. This prompted me to log in the log and I can see that I get the error of the type:
core.conformation.carbohydrates.util: [ ERROR ] get_non_NU_TorsionID_from_AtomIDs() could not determine the BB id for theTorsionID defined by these atoms: atomno= 5 rsd= 10 ; atomno= 6 rsd= 10 ; atomno= 8 rsd= 10 ; atomno= 1 rsd= 56
core.conformation.carbohydrates.util: [ ERROR ] get_non_NU_TorsionID_from_AtomIDs() could not determine the BB id for theTorsionID defined by these atoms: atomno= 5 rsd= 10 ; atomno= 6 rsd= 10 ; atomno= 8 rsd= 10 ; atomno= 1 rsd= 56
core.conformation.carbohydrates.util: [ ERROR ] get_non_NU_TorsionID_from_AtomIDs() could not determine the BB id for theTorsionID defined by these atoms: atomno= 5 rsd= 42 ; atomno= 6 rsd= 42 ; atomno= 7 rsd= 42 ; atomno= 1 rsd= 59
core.conformation.carbohydrates.util: [ ERROR ] get_non_NU_TorsionID_from_AtomIDs() could not determine the BB id for theTorsionID defined by these atoms: atomno= 5 rsd= 45 ; atomno= 6 rsd= 45 ; atomno= 7 rsd= 45 ; atomno= 1 rsd= 60
core.conformation.carbohydrates.util: [ ERROR ] get_non_NU_TorsionID_from_AtomIDs() could not determine the BB id for theTorsionID defined by these atoms: atomno= 5 rsd= 48 ; atomno= 6 rsd= 48 ; atomno= 7 rsd= 48 ; atomno= 1 rsd= 61Interestingly, it is core.conformation.carbohydrates.util which outputs the error, but the errors are printed after GlycanTreeModeler has printed the summary of its run e.g.
protocols.carbohydrates.GlycanTreeModeler: Start- : 13198.6
protocols.carbohydrates.GlycanTreeModeler: Pre - : 790.491
protocols.carbohydrates.GlycanTreeModeler: Post - : 829.677
protocols.carbohydrates.GlycanTreeModeler: Final- : 790.491
core.conformation.carbohydrates.util: [ ERROR ] get_non_NU_TorsionID_from_AtomIDs() could not determine the BB id for theTorsionID defined by these atoms: atomno= 5 rsd= 4 ; atomno= 6 rsd= 4 ; atomno= 8 rsd= 4 ; atomno= 1 rsd= 53
core.conformation.carbohydrates.util: [ ERROR ] get_non_NU_TorsionID_from_AtomIDs() could not determine the BB id for theTorsionID defined by these atoms: atomno= 5 rsd= 10 ; atomno= 6 rsd= 10 ; atomno= 8 rsd= 10 ; atomno= 1 rsd= 56
core.conformation.carbohydrates.util: [ ERROR ] get_non_NU_TorsionID_from_AtomIDs() could not determine the BB id for theTorsionID defined by these atoms: atomno= 5 rsd= 42 ; atomno= 6 rsd= 42 ; atomno= 7 rsd= 42 ; atomno= 1 rsd= 59
...One thing I learned is that if I output a pose (pose.dump_pdb) and reload it again, it get the following warnings which are likely connected to the AtomID errors:
core.io.pose_from_sfr.PoseFromSFRBuilder: [ WARNING ] Glc53 has an unfavorable ring conformation; the coordinates for this input structure may have been poorly assigned.
core.io.pose_from_sfr.PoseFromSFRBuilder: [ WARNING ] Gal54 has an unfavorable ring conformation; the coordinates for this input structure may have been poorly assigned.
core.io.pose_from_sfr.PoseFromSFRBuilder: [ WARNING ] Neu55 has an unfavorable ring conformation; the coordinates for this input structure may have been poorly assigned.
core.io.pose_from_sfr.PoseFromSFRBuilder: [ WARNING ] Glc56 has an unfavorable ring conformation; the coordinates for this input structure may have been poorly assigned.
...
core.chemical.AtomICoor: [ WARNING ] IcoorAtomID::atom_id(): Cannot get atom_id for POLYMER_LOWER of residue ->4)-beta-D-Glcp:2-AcNH 53. Returning BOGUS ID instead.
core.chemical.AtomICoor: [ WARNING ] IcoorAtomID::atom_id(): Cannot get atom_id for POLYMER_LOWER of residue ->4)-beta-D-Glcp:2-AcNH 56. Returning BOGUS ID instead.
core.chemical.AtomICoor: [ WARNING ] IcoorAtomID::atom_id(): Cannot get atom_id for POLYMER_LOWER of residue ->4)-alpha-D-Manp:non-reducing_end 59. Returning BOGUS ID instead.
core.chemical.AtomICoor: [ WARNING ] IcoorAtomID::atom_id(): Cannot get atom_id for POLYMER_LOWER of residue ->4)-alpha-D-Manp:non-reducing_end 60. Returning BOGUS ID instead.
core.chemical.AtomICoor: [ WARNING ] IcoorAtomID::atom_id(): Cannot get atom_id for POLYMER_LOWER of residue ->4)-alpha-D-Manp:non-reducing_end 61. Returning BOGUS ID instead.
core.chemical.AtomICoor: [ WARNING ] IcoorAtomID::atom_id(): Cannot get atom_id for POLYMER_LOWER of residue ->3)-alpha-L-Fucp 62. Returning BOGUS ID instead.
core.chemical.AtomICoor: [ WARNING ] IcoorAtomID::atom_id(): Cannot get atom_id for POLYMER_LOWER of residue ->3)-alpha-L-Fucp 64. Returning BOGUS ID instead.In conclusion:
I get both warning, error and failure flags whenever I try to add glycosylations. If I furthermore try to use other movers to perturb the backbone in hinge regions, no moves are accepted. Here is a dummy version of the script where I add the glycosylation to a AA sequence I just cooked up, in case it has something to do with the way I do it:
from pyrosetta import *
pyrosetta.init('-include_sugars','-write_pdb_link_records')#,'-auto_detect_glycan_connections')
#%%
from pyrosetta.rosetta.protocols import relax
from pyrosetta.rosetta.protocols.simple_moves import SmallMover, ShearMover
from pyrosetta.rosetta.protocols.minimization_packing import MinMover, PackRotamersMover
from pyrosetta.rosetta.core.pack.task import TaskFactory
from pyrosetta.rosetta.core.pose.carbohydrates import glycosylate_pose
from pyrosetta.rosetta.core.pose import make_pose_from_saccharide_sequence,
pose_from_saccharide_sequence
from pyrosetta.rosetta.protocols.carbohydrates import *
from pyrosetta.rosetta.core.pose.carbohydrates import glycosylate_pose, glycosylate_pose_by_file
from pyrosetta.rosetta.utility import vector1_std_string as vstr
sugar_bb = pyrosetta.rosetta.core.scoring.ScoreType.sugar_bb
#%%
import os, glob
import numpy as np
from timeit import default_timer as timer
import datetime
#%%
path = os.path.abspath('.') + '/'
os.chdir(path)
sequence = 'NWS'*4+'PPPP'*2+'NWS'*4+'PPPP'*2+'AWA'*4
pose = pose_from_sequence(sequence)
#pose.dump_pdb('NoPTM.pdb')
#%%
string1 = '->-a-Neup5Ac-(2->3)-b-D-Galp-(1->4)-b-D-GlcpNAc-(1->2)-a-D-Manp-(1->6)-'+
'[->-a-Neup5Ac-(2->3)-b-D-Galp-(1->4)-b-D-GlcpNAc-(1->2)-a-D-Manp-(1->3)]-' +
'[b-D-GlcpNAc-(1->4)]-b-D-Manp-(1->4)-b-D-GlcpNAc-(1->4)-'+
'[a-L-Fucp-(1->6)]-b-D-GlcpNAc'
string2 = 'b-D-Glcp-(1->3)-a-L-Fucp'
string3 = '->4)-a-D-Manp'
#%%
string1 = '->-a-Neup5Ac-(2->3)-b-D-Galp-(1->4)-b-D-GlcpNAc-(1->2)-a-D-Manp-(1->6)-'+
'[->-a-Neup5Ac-(2->3)-b-D-Galp-(1->4)-b-D-GlcpNAc-(1->2)-a-D-Manp-(1->3)]-' +
'[b-D-GlcpNAc-(1->4)]-b-D-Manp-(1->4)-b-D-GlcpNAc-(1->4)-'+
'[a-L-Fucp-(1->6)]-b-D-GlcpNAc'
string2 = 'b-D-Glcp-(1->3)-a-L-Fucp'
string3 = '->4)-a-D-Manp'
#%%
mov = SimpleGlycosylateMover()
mov.set_glycosylation(string1)
pos = [4,10]
for ps in pos:
mov.set_position(ps)
mov.apply(pose)
mov.set_glycosylation(string3)
pos = [42,45,48]
for ps in pos:
mov.set_position(ps)
mov.apply(pose)
mov.set_glycosylation(string2) # cannot finish with mannose addition, throws error
pos = [23,26]
for ps in pos:
mov.set_position(ps)
mov.apply(pose)
#%%
scorefxn = get_fa_scorefxn()
kT = 2
n_moves = 1
mc = MonteCarlo(pose, scorefxn, kT)
# Glycosylation mins
mins_glyc = GlycanTreeModeler()
# prepare for hinge region
hinge_reg = [1,52]
hinge = MoveMap()
hinge.set_bb_true_range(hinge_reg[0], hinge_reg[1])
# Movers for hinge region
smallmover = SmallMover(hinge, kT, n_moves)
smallmover.angle_max(10)
shearmover = ShearMover(hinge, kT, n_moves)
# optimese rotamers
to_pack = TaskFactory.create_packer_task(pose)
to_pack.restrict_to_repacking() # prevents design, packing only
to_pack.or_include_current(True)
packmover = PackRotamersMover(scorefxn, to_pack)
# prepare the sequence mover
seq_mover = SequenceMover()
seq_mover.add_mover(mins_glyc)
num_reps = 10 #
for num in range(num_reps):
seq_mover.add_mover(smallmover)
seq_mover.add_mover(shearmover)
seq_mover.add_mover(packmover)
trial_mover = TrialMover(seq_mover, mc)
file = 'Dummy.log'
header = '# kT = {:0.0f}, n moves/mover = {:d}n'.format(kT, n_moves) +
'# Timestamp, elapsed, Cycle, Score [REU], Acc. rate [Acc/trials]n'
with open(file,'w+') as f:
f.writelines(header)
time = timer()
for num in range(200):
trial_mover.apply(pose)
if np.mod(num+1,1) == 0: # conditional used if I want to output less
E_temp = scorefxn(pose)
elapsed = timer()-time
date = datetime.datetime.now().strftime("%H:%M:%S")
acc_rate = trial_mover.acceptance_rate()
string = date + ', {:0.0f}, {:d}'.format(elapsed, num+1) +
', {:0.2f}, {:0.2e}n'.format(E_temp, acc_rate)
f.writelines(string)
if np.mod(num+1,1) == 0: # conditional used if I want to output less
filename = 'dum_{:03d}.pdb'.format(num+1)
pose.dump_pdb(filename)
void = mc.recover_low(pose)
filename = 'xDum_lowest.pdb'
pose.dump_pdb(filename)
Note that in this script, the movers are able to change BB angles
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