Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand
Hi Nestor, I think the reason for Arg side chain to curl up is because I refined ligand and Arg side chain with occupancy refinement, and the Arg moved away from the density, most likely due to 'repulsion' from ligand. The other question is: Is it possible that the H-bonds stay very close? As I tried to 'real space refine' in coot, and the Arg side chain flipped away from the density. Thanks for the suggestion. Best HK Quoting Nestor Concha : Hi Tam, The density looks very strong and therefore I'm going to guess that the Arg guanidimium stays in contact/interacts with the ligand and with the phosphate/sulfate next to it. Perhaps it is one of those close interactions with shorter H-bonds that usual given the arrangement of ligand-phosphate/sulfate-Arg. I'd try to find a rotamer for the Arg that leaves the interactions intact rather than refine occupancies. Seems that the Arg side chain is curled up Nestor -Original Message- From: CCP4 bulletin board On Behalf Of Heng-Keat Tam Sent: Tuesday, November 5, 2019 10:55 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand EXTERNAL Dear Rob, I would like to model the alternative position for the side chain of R120 but I don't really know whether the alternative conformation exist as shown in the attached figure - density without the ligand and R120 overlaid with the refined structures of modeled ligand and R120. The ligand was modeled in two different conformations. From the density, it seems to me that the density is connected or overlapped. It should not be a post-translation modification as it is well-known that there is no post-translation modification for this protein. Furthermore, the crystal was obtained by co-crystallization of protein with the ligand itself. The density seems to be the expected ligand. Thanks for the advice. Best regards HK Quoting Robert Nicholls : Dear HK, No that's not quite correct - 'occupancy group alts complete' means that both R120 and the ligand are constrained so that their occupancies sum to unity. In contrast, 'occupancy group alts incomplete' means that the occupancies of R120 and the ligand will not be constrained to sum to unity (but the sum of their occupancies must be less than one). In both cases, R120 and the ligand will "see" each other in a certain sense. But, because they are assigned to different groups, it is assumed that they are present in different parts of the crystal. This means that they can overlap. Assuming that the ligand is in the correct conformation, I suspect the source of your problem is that you are modelling the side chain of R120 as only one conformation. And I would also include the other atoms in the side chain - CB and CG. If you are modelling the sidechain of R120 with partial occupancies, then you should model those side chain atoms in two alternative positions (i.e. representing the portions of the crystal that do/don't have the ligand bound). This will help to ensure that your model makes physical sense. So the ligand plus the alt of R120 in the portion of the crystal that contains the ligand would be assigned to one occupancy group, and the alt of R120 in the portion of the crystal that does not contain the ligand would be assigned to the second group. In this case it would be appropriate to specify 'occupancy group alts complete', because the occupancies for the two alternative conformers of R120 should sum to unity. Although no doubt the estimation of the occupancies would be dominated by the ligand in this case. Be sure to check your B-factors after occupancy refinement to make sure the whole picture makes sense. Assuming your current model is essentially correct, from visual inspection it looks like R120 will have low occupancy and low B-factors when the ligand is not present (or at least B-factors consistent with the environment), but will have high occupancy and high B-factors when the ligand is present. On another note, have you considered whether that part of the ligand could be modelled in a slightly different conformation, or whether there could be a post-translation modification? I hope that helps, Rob Dr Rob Nicholls Senior Investigator Scientist MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH On 5 Nov 2019, at 13:36, HK wrote: Dear all, I have problem with occupancy refinement by Refmac5 for an overlapping electron density of part of residue (an arginine) and part of ligand (tetracyclic compound) (attached figures in Dropbox with a link as shown below). https://www.dropbox.com/sh/ppmfp5dnpy1b9e9/AAAV79bOzPHQUrVYp9loMwyha? dl=0 I refined part of the side chain of residue 120 and ligand (chain J residue 1105) with Refmac keyword as shown below. Unfortunately, the side chain of arginine moves away from the density but the ligand stays in the density. As far as I
[ccp4bb] [Second Notice] BioSAXS2019 workshop at SSRL/SLAC
Second Notice: Dear all, We are glad to announce the BioSAXS2019, a workshop on biological small-angle X-ray scattering at Stanford Synchrotron Radiation Lightsource (SSRL). When: December 2-5, 2019 Where: SSRL, SLAC National Accelerator Laboratory, Menlo Park, CA The SSRL Structural Molecular Biology Group hosts a 4-day comprehensive workshop on the use of non-crystalline small-angle x-ray scattering and diffraction techniques in structural biology research. The workshop will focus on solution x-ray scattering studies on biological macromolecules and macromolecular complexes and cover the basic theory of small angle scattering, experimental aspects of solution scattering as well as recent applications of solution scattering in structural biology research. There will be extensive software and data analysis tutorials covering all aspects from the basic analysis of SAXS data to advanced modeling methods. Participants are encouraged to bring their own solution samples for the data collection tutorial at SSRL Beam Line 4-2. Attendance is limited so register early to save your spot. Check out the website: https://conf.slac.stanford.edu/smbsax2019/ for more details on the program and registration. Please feel free to contact me if you have any question, etc. Tsutomu Matsui -- *** Tsutomu Matsui, D.Sc. Stanford Synchrotron Radiation Lightsource (SSRL) 2575 Sand Hill Rd, MS69, Menlo Park, CA 94025 TEL: 650-926-5598 FAX: 650-926-4100 *** To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Fwd: CCP4i2 London Road Show
Reminder One week to London CCP4i2 Road show. Places still available. *CCP4 i2 Road Show, London* There will be a CCP4 Road Show for use of the i2 interface on Tuesday 12th November, at Birkbeck College. *Time:* 14:00 - 16:00 for the main workshop with about an hour afterwards for those with additional questions. *Place:* Room 416 in the main Birkbeck Malet St Building. There will be 2-3 CCP4 tutors presenting the Road Show. While it is primarily intended for the extensive number of crystallographers in London, all are welcome. There will be places for 40 participants. Desktops will be used, but you are also welcome to use your laptops (but if doing so please ensure you install the software in advance - see CCP4 webpages for guidance on how to do this). To book a place on a first come first served basis please register at http://www.bbk.ac.uk/booking/event/8829 Event page is http://www.bbk.ac.uk/events/remote_event_view?id=8829 Best wishes Nick Keep and Keith Wilson -- Prof Nicholas H. Keep Executive Dean of School of Science Professor of Biomolecular Science Crystallography, Institute for Structural and Molecular Biology, Department of Biological Sciences Birkbeck, University of London, Malet Street, Bloomsbury LONDON WC1E 7HX emailn.k...@mail.cryst.bbk.ac.uk Telephone 020-7631-6852 (Room G54a Office) 020-7631-6800 (Department Office) Fax 020-7631-6803 If you want to access me in person you have to come to the crystallography entrance and ring me or the department office from the internal phone by the door To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] lysine methylation in E. coli?
It happens. Ef-Tu is methylated on a single lysine Also LafV is a putative methylase in E. Coli but its effects were not studied in detail. Salmonella methylates flagellin which is how lysine methylation was discovered - and E.coli is a kissing cousin so... Artem On Tue, Nov 5, 2019, 12:31 Jennifer Fleming wrote: > > Dear CCP4 community, > > > > I am hoping that someone here might know the answer to this as I cannot > find anything concrete in my literature search. > > > > I have recently received a mass spec result indicating that a recombinant > protein expressed in *E. coli* is heavily lysine methylated. I would like > to know is this an artefact and is it even possible for *E. coli* to > methylate lysines? > > > > Thank you for any help, > > > > Jen > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] lysine methylation in E. coli?
Dear CCP4 community, I am hoping that someone here might know the answer to this as I cannot find anything concrete in my literature search. I have recently received a mass spec result indicating that a recombinant protein expressed in /E. coli/ is heavily lysine methylated. I would like to know is this an artefact and is it even possible for /E. coli/ to methylate lysines? Thank you for any help, Jen To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand
Dear HK, If you believe that R120 only adopts one conformation then you should not refine the occupancies of the side chain atoms. It makes no physical sense to have occupancies less than one for these atoms, but occupancy equal to one for the rest of the protein (unless you believe the side chain to be cleaved, or otherwise chemically modified). However, occupancy refinement of the ligand itself can be justified by partial binding. Best regards, Rob > On 5 Nov 2019, at 15:55, t...@em.uni-frankfurt.de wrote: > > Dear Rob, > > I would like to model the alternative position for the side chain of R120 but > I don't really know whether the alternative conformation exist as shown in > the attached figure - density without the ligand and R120 overlaid with the > refined structures of modeled ligand and R120. The ligand was modeled in two > different conformations. From the density, it seems to me that the density is > connected or overlapped. > > It should not be a post-translation modification as it is well-known that > there is no post-translation modification for this protein. Furthermore, the > crystal was obtained by co-crystallization of protein with the ligand itself. > The density seems to be the expected ligand. > > Thanks for the advice. > > Best regards > HK > > > Quoting Robert Nicholls : > >> Dear HK, >> >> No that's not quite correct - 'occupancy group alts complete' means that >> both R120 and the ligand are constrained so that their occupancies sum to >> unity. In contrast, 'occupancy group alts incomplete' means that the >> occupancies of R120 and the ligand will not be constrained to sum to unity >> (but the sum of their occupancies must be less than one). In both cases, >> R120 and the ligand will "see" each other in a certain sense. But, because >> they are assigned to different groups, it is assumed that they are present >> in different parts of the crystal. This means that they can overlap. >> >> Assuming that the ligand is in the correct conformation, I suspect the >> source of your problem is that you are modelling the side chain of R120 as >> only one conformation. And I would also include the other atoms in the side >> chain - CB and CG. >> >> If you are modelling the sidechain of R120 with partial occupancies, then >> you should model those side chain atoms in two alternative positions (i.e. >> representing the portions of the crystal that do/don't have the ligand >> bound). This will help to ensure that your model makes physical sense. So >> the ligand plus the alt of R120 in the portion of the crystal that contains >> the ligand would be assigned to one occupancy group, and the alt of R120 in >> the portion of the crystal that does not contain the ligand would be >> assigned to the second group. In this case it would be appropriate to >> specify 'occupancy group alts complete', because the occupancies for the two >> alternative conformers of R120 should sum to unity. Although no doubt the >> estimation of the occupancies would be dominated by the ligand in this case. >> >> Be sure to check your B-factors after occupancy refinement to make sure the >> whole picture makes sense. Assuming your current model is essentially >> correct, from visual inspection it looks like R120 will have low occupancy >> and low B-factors when the ligand is not present (or at least B-factors >> consistent with the environment), but will have high occupancy and high >> B-factors when the ligand is present. >> >> On another note, have you considered whether that part of the ligand could >> be modelled in a slightly different conformation, or whether there could be >> a post-translation modification? >> >> I hope that helps, >> Rob >> >> >> Dr Rob Nicholls >> Senior Investigator Scientist >> MRC Laboratory of Molecular Biology >> Francis Crick Avenue >> Cambridge Biomedical Campus >> Cambridge CB2 0QH >> >> >> >>> On 5 Nov 2019, at 13:36, HK wrote: >>> >>> Dear all, >>> >>> I have problem with occupancy refinement by Refmac5 for an overlapping >>> electron density of part of residue (an arginine) and part of ligand >>> (tetracyclic compound) (attached figures in Dropbox with a link as shown >>> below). >>> >>> https://www.dropbox.com/sh/ppmfp5dnpy1b9e9/AAAV79bOzPHQUrVYp9loMwyha?dl=0 >>> >>> I refined part of the side chain of residue 120 and ligand (chain J residue >>> 1105) with Refmac keyword as shown below. Unfortunately, the side chain of >>> arginine moves away from the density but the ligand stays in the density. >>> As far as I understood, occupancy refinement with keyword 'occupancy group >>> alts complete' means both R120 and the ligand do not meet each other. Did >>> I miss something from the occupancy refinement keyword? >>> >>> occupancy group id 1 chain A residue 120 atom NE >>> occupancy group id 1 chain A residue 120 atom CZ >>> occupancy group id 1 chain A residue 120 atom NH2 >>> occupancy group id 1 chain A
[ccp4bb] Postdoctoral position available at the Basque Centre for Biophysics (Bilbao, SPAIN)
Dear colleagues, We are currently looking for a postdoctoral researcher to join our lab. Please see below for details: *OFFER* – Postdoctoral Position (Two years) *DEADLINE: *4th of February, 2020 at 17:00 h CET or until position is filled. *WHERE* – Basque Centre for Biophysics (Bilbao, SPAIN) The Basque Centre for Biophysics is an international research center of excellence with the main aim of promoting a multidisciplinary research program in the field of Biophysics and its application in the areas of Biotechnology and Health, the center is a joint effort of the University of the Basque Country (UPV/EHU) and the Spanish National Research Council (CSIC). *Description of the position offered* The position is available in the Structural Microbiology Laboratory led by Dr. David Albesa-Jové at the Basque Centre for Biophysics (Bilbao, Spain). Our group was recently established and focuses on understanding the biological function of virulence factors and essential enzymes in human pathogenic bacteria. Please, see below for some recent publications. The laboratory is well equipped for all aspects of molecular biology, biochemistry, biophysics and crystallography. We also have regular access to synchrotron radiation at Diamond (UK) and ALBA (Spain) through BAG applications. 1.*Albesa-Jové** D, et al.: Structural Snapshots of α-1,3-Galactosyltransferase with Native Substrates: Insight into the Catalytic Mechanism of Retaining Glycosyltransferases. *Angew Chemie Int Ed* 2017, 56:14853–14857. *IF. 12.26 *corresponding author* 2.Planamente S, Salih O, Manoli E, *Albesa**‐**Jové D*, Freemont PS, Filloux A: TssA forms a gp6‐like ring attached to the type VI secretion sheath. *EMBO J.* 2016, 35:1613–1627. *IF. 11.23* 3.*Albesa-Jové *D, et al.: Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA *Nature Communications*. 2016, 7:10906. *IF. 11.89* 4.*Albesa-Jové* D, et al.: A native ternary complex in crystal reveals the catalytic mechanism of a retaining glycosyltransferase. *Angew Chem Int Ed Engl.* 2015; 54(34): 9898-902. *IF. 12.26* 5.Giganti D, *Albesa-Jové* D, et al.: Secondary structure reshuffling modulates glycosyltransferase function at the membrane. *Nature Chemical Biology.* 2015, 11(1): 16–18. *IF. 12.15* 6.Andrés A1, *Albesa-Jové1* D, et al.: Structural basis of chitin oligosaccharide deacetylation. *Angew Chem Int Ed Engl.* 2014, 53(27): 6882–6887. *IF. 12.26 1equal contribution* *Education and Experience Required* We are seeking a highly motivated individual with a strong background in structural biology, including protein expression and purification, protein crystallization, and X-ray crystallography. Outstanding postdoctoral candidates will also have the necessary support to apply for the Ikerbasque Fellows 2020 call. These 5-year Fellowships are directed to promising young postdoctoral researchers (applicants must have their PhD completed between 2009 and 2017) and are intended to offer a track towards independent research as a group leader. Ikerbasque Fellows in their 5th year can be assessed for permanent group leader positions. More details about the previous 2019 Ikerbasque Fellows call can be found here: http://www.ikerbasque.net/sites/default/files/files/Ikerbasque_RF_2019_Call_Specifications.pdf *Contact:* Please submit your application for the postdoctoral position through the Centre’s website (http://biofisika.org/contact/) indicating the following subject: *Job Application: 52Albesa*, and adding a cover letter, curriculum vitae, and contact information for three professional referees. Informal enquires can be directed to davidalb...@gmail.com. Women are encouraged to apply. == David Albesa-Jové, Ph.D. Ikerbasque Research Associate Head of the Structural Microbiology Laboratory Instituto Biofísika (CSIC-UPV/EHU) Scientific Park of the University of the Basque Country Barrio Sarriena s/n, 48940 Leioa SPAIN Tel – office: +34 94 601 5171 Tel – lab: +34 94 601 3356 http://www.researcherid.com/rid/F-5683-2016 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand
Dear HK, No that's not quite correct - 'occupancy group alts complete' means that both R120 and the ligand are constrained so that their occupancies sum to unity. In contrast, 'occupancy group alts incomplete' means that the occupancies of R120 and the ligand will not be constrained to sum to unity (but the sum of their occupancies must be less than one). In both cases, R120 and the ligand will "see" each other in a certain sense. But, because they are assigned to different groups, it is assumed that they are present in different parts of the crystal. This means that they can overlap. Assuming that the ligand is in the correct conformation, I suspect the source of your problem is that you are modelling the side chain of R120 as only one conformation. And I would also include the other atoms in the side chain - CB and CG. If you are modelling the sidechain of R120 with partial occupancies, then you should model those side chain atoms in two alternative positions (i.e. representing the portions of the crystal that do/don't have the ligand bound). This will help to ensure that your model makes physical sense. So the ligand plus the alt of R120 in the portion of the crystal that contains the ligand would be assigned to one occupancy group, and the alt of R120 in the portion of the crystal that does not contain the ligand would be assigned to the second group. In this case it would be appropriate to specify 'occupancy group alts complete', because the occupancies for the two alternative conformers of R120 should sum to unity. Although no doubt the estimation of the occupancies would be dominated by the ligand in this case. Be sure to check your B-factors after occupancy refinement to make sure the whole picture makes sense. Assuming your current model is essentially correct, from visual inspection it looks like R120 will have low occupancy and low B-factors when the ligand is not present (or at least B-factors consistent with the environment), but will have high occupancy and high B-factors when the ligand is present. On another note, have you considered whether that part of the ligand could be modelled in a slightly different conformation, or whether there could be a post-translation modification? I hope that helps, Rob Dr Rob Nicholls Senior Investigator Scientist MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH > On 5 Nov 2019, at 13:36, HK wrote: > > Dear all, > > I have problem with occupancy refinement by Refmac5 for an overlapping > electron density of part of residue (an arginine) and part of ligand > (tetracyclic compound) (attached figures in Dropbox with a link as shown > below). > > https://www.dropbox.com/sh/ppmfp5dnpy1b9e9/AAAV79bOzPHQUrVYp9loMwyha?dl=0 > > I refined part of the side chain of residue 120 and ligand (chain J residue > 1105) with Refmac keyword as shown below. Unfortunately, the side chain of > arginine moves away from the density but the ligand stays in the density. As > far as I understood, occupancy refinement with keyword 'occupancy group alts > complete' means both R120 and the ligand do not meet each other. Did I miss > something from the occupancy refinement keyword? > > occupancy group id 1 chain A residue 120 atom NE > occupancy group id 1 chain A residue 120 atom CZ > occupancy group id 1 chain A residue 120 atom NH2 > occupancy group id 1 chain A residue 120 atom NH1 > occupancy group id 1 chain A residue 120 atom CD > occupancy group id 2 chain J residue 1105 > occupancy group alts complete 1 2 > occupancy refine > > Thank you for the advice. > > Best regards > HK > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand
Dear all, I have problem with occupancy refinement by Refmac5 for an overlapping electron density of part of residue (an arginine) and part of ligand (tetracyclic compound) (attached figures in Dropbox with a link as shown below). https://www.dropbox.com/sh/ppmfp5dnpy1b9e9/AAAV79bOzPHQUrVYp9loMwyha?dl=0 I refined part of the side chain of residue 120 and ligand (chain J residue 1105) with Refmac keyword as shown below. Unfortunately, the side chain of arginine moves away from the density but the ligand stays in the density. As far as I understood, occupancy refinement with keyword 'occupancy group alts complete' means both R120 and the ligand do not meet each other. Did I miss something from the occupancy refinement keyword? occupancy group id 1 chain A residue 120 atom NE occupancy group id 1 chain A residue 120 atom CZ occupancy group id 1 chain A residue 120 atom NH2 occupancy group id 1 chain A residue 120 atom NH1 occupancy group id 1 chain A residue 120 atom CD occupancy group id 2 chain J residue 1105 occupancy group alts complete 1 2 occupancy refine Thank you for the advice. Best regards HK To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Introducing Dials User Interface for CCP4
On 05/11/2019 09:53, Fuentes-Montero, Luis (DLSLtd,RAL,LSCI) wrote: Hi FH, It looks to me like an installation issue, I am CCing you to the rest of the CCP4BB with hope to find anyone who knows what to do: Guys: Does anybody faced the following error message before? *undefined symbol: PyUnicodeUCS2_DecodeUTF8* Thanks in advance, I have an error when runnig DUI with las version of dials (14.12) _ undefined symbol: PyUnicodeUCS2_DecodeUTF8 What's the problem ?? Compile python with --enable-unicode=ucs2 Paul. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Introducing Dials User Interface for CCP4
Hi FH, It looks to me like an installation issue, I am CCing you to the rest of the CCP4BB with hope to find anyone who knows what to do: Guys: Does anybody faced the following error message before? undefined symbol: PyUnicodeUCS2_DecodeUTF8 Thanks in advance, Luiso. From: hoh Sent: 04 November 2019 16:23 To: Fuentes-Montero, Luis (DLSLtd,RAL,LSCI) Subject: Re: [ccp4bb] Introducing Dials User Interface for CCP4 Hi Luis I have an error when runnig DUI with las version of dials (14.12) _ undefined symbol: PyUnicodeUCS2_DecodeUTF8 What's the problem ?? Thank's FH ___ François Hoh Centre de Biochimie Structurale, UMR 5048 CNRS, UMR 1054 INSERM 29, rue de navacelles 34090 Montpellier Cedex, France. Phone: +33 467 417 706 Fax: +33 467 417 913 -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] why does Coot ignore CONECT?
Hi Pavel I work with peptide-scaffold chimeras on a daily basis. Initially, I tried to include the scaffolds as HETATM and use JLigand to create the links between the scaffolds and the natural amino acids. This procedure however is rather fussy and by now I build the chimera in silico, using natural amino acids provided by the program. Subsequently, I run the energy minimization on my own to have a rough control over the conformation. I then feed the coordinates into PRODRG without any further minimization and use the pdb and the restraints as they come from the server. Of course, such a ligand will not make use of the cif libraries of the natural amino acids during refinement. But if you imported the natural amino acids while building the chimera, your minimization used the proper restraints. I have the impression that this procedure is more efficient than trying to maintain the LINK in the pdb header (which, in my case, oftern got lost after running a refinement, so I had to copy-paste it anew after every cycle). Best, Matthias Dr. Matthias Barone AG Kuehne, Rational Drug Design Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Strasse 10 13125 Berlin Germany Phone: +49 (0)30 94793-284 From: CCP4 bulletin board on behalf of Pavel Mader Sent: Monday, November 4, 2019 6:06:11 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] why does Coot ignore CONECT? Hi Paul, thank you for your answer. My problem is that I am building a cyclic peptide with non-standard amino acids, the side chains of which are linked by click chemistry... I know how to make modified amino acids that will make peptide bonds with their neighbors in the polypeptide chain, but I don't know how to make a covalent link between side chains of amino acids say No. 3 and 12 for example (of a 15 AA long chain). I was trying to solve the issue by making a CONECT record in the pdb file, but Coot ignored it. LINK "sort of" worked, but it was not possible to control the geometry (bond length and angles), often the real space refinement simply "explodes" probably in a attempt to avoid clashes. My current workaround is to make a cif file for the whole 15 AA long polypeptide (the bonds and angles now behave more or less as expected), but I don't consider this as a good general practice for building long polypeptide chains... Thanks in advance for any hints guiding me in the right direction. Pavel -- Původní e-mail -- Od: Paul Emsley Komu: CCP4BB@JISCMAIL.AC.UK Datum: 1. 11. 2019 21:16:11 Předmět: Re: [ccp4bb] why does Coot ignore CONECT? On 01/11/2019 20:17, Pavel Mader wrote: > Hello, Hello. > > I have a question, can anyone explain, why does Coot not display a covalent > bond manually specified by > CONECT line in a pdb file? Because I have never thought them necessary. Using SSBOND, LINK and residue dictionaries seemed to me to cover the bases for which CONECT would be used. Paul. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Unusual dataset with high Rmerge and extremely low b-factors?
Hi Michael, I believe you are looking at the anisotropic B factor. 8 A2 is very low, you are all good. The Wilson B is given by Truncate if I'm not mistaking. Best regards Vincent Le 04/11/2019 à 00:18, Michael Jarva a écrit : Hi CCP4BB, I have some unusual crystal diffraction data I'd like to get your input on. Almost a year ago I shot some small rods sticking out of a loop, so basically no liquid around them - using the microfocus MX2 beamline at the australian synchrotron, collected on an EIGER 16M detector. The crystals diffracted weakly and was seemingly not viable at first glance because of high Rmerge/Rpims. See the aimless summary at the bottom of this post. This seemed to stem from a low spot intensity at low resolutions (I/sd(I)=4.6), but since the CC1/2 was fine I went with it anyway. Here I also noted an unusually low Mosaicity, 0.05, and Wilson B-factors, 8.02 Å^2. Density maps looked great and the build refined easily enough (R/Rfree 0.1939/0.2259) with a mean B-factor of 19.85, which according to phenix is lower than any other structure deposited in that resolution bin. Furthermore, the molprobity score is 0.83, and overall real-space correlation CC is 0.855. So my question is, can I feel comfortable depositing this? best regards Michael Chosen Solution: space group P 1 21 1 Unit cell: 44.93 41.90 45.83 90.00 115.57 90.00 Number of batches in file: 1659 The data do not appear to be twinned, from the L-test Overall InnerShell OuterShell Low resolution limit 41.34 41.34 2.49 High resolution limit 2.40 8.98 2.40 Rmerge (within I+/I-) 0.231 0.084 0.782 Rmerge (all I+ and I-) 0.266 0.099 0.983 Rmeas (within I+/I-) 0.323 0.118 1.091 Rmeas (all I+ & I-) 0.317 0.118 1.167 Rpim (within I+/I-) 0.225 0.084 0.759 Rpim (all I+ & I-) 0.171 0.063 0.623 Rmerge in top intensity bin 0.079 - - Total number of observations 19901 362 2067 Total number unique 6054 126 611 Mean((I)/sd(I)) 2.7 4.6 1.0 Mn(I) half-set correlation CC(1/2) 0.958 0.991 0.570 Completeness 98.6 98.2 97.3 Multiplicity 3.3 2.9 3.4 Mean(Chi^2) 0.48 0.33 0.50 Anomalous completeness 81.7 92.2 75.1 Anomalous multiplicity 1.5 1.8 1.9 DelAnom correlation between half-sets -0.003 0.041 0.045 Mid-Slope of Anom Normal Probability 0.704 - - The anomalous signal appears to be weak so anomalous flag was left OFF Estimates of resolution limits: overall from half-dataset correlation CC(1/2) > 0.30: limit = 2.40A == maximum resolution from Mn(I/sd) > 1.50: limit = 2.67A from Mn(I/sd) > 2.00: limit = 2.87A Estimates of resolution limits in reciprocal lattice directions: Along 0.96 a* - 0.28 c* from half-dataset correlation CC(1/2) > 0.30: limit = 2.40A == maximum resolution from Mn(I/sd) > 1.50: limit = 2.40A == maximum resolution Along k axis from half-dataset correlation CC(1/2) > 0.30: limit = 2.40A == maximum resolution from Mn(I/sd) > 1.50: limit = 2.86A Along -0.17 a* + 0.99 c* from half-dataset correlation CC(1/2) > 0.30: limit = 2.40A == maximum resolution from Mn(I/sd) > 1.50: limit = 2.98A Anisotropic deltaB (i.e. range of principal components), A^2: 8.62 Average unit cell: 44.93 41.90 45.83 90.00 115.57 90.00 Space group: P 1 21 1 Average mosaicity: 0.05 Minimum and maximum SD correction factors: Fulls 1.27 1.28 Partials 0.00 0.00 Michael Jarva, PhD ACRF Chemical Biology Division The Walter and Eliza Hall Institute of Medical Research 1G Royal Parade Parkville Victoria 3052 Australia Phone: +61 3 9345 2493 Email: jarv...@wehi.edu.au | Web: http://www.wehi.edu.au/ The ACRF Chemical Biology Division is supported by the Australian Cancer Research Foundation ___ The information in this email is confidential and intended solely for the addressee. You must not disclose, forward, print or use it without the permission of the sender. The Walter and Eliza Hall Institute acknowledges the Wurundjeri people of the Kulin Nation as the traditional owners of the land where our campuses are located and the continuing connection to country and community. ___ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 -- Vincent Chaptal, PhD MMSB -UMR5086 Drug Resistance and Membrane Proteins Laboratory 7 passage du Vercors 69007 LYON FRANCE +33 4 37 65 29 01 http://mmsb.cnrs.fr/en/ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
