Dear All, Thank you very much for your valued suggestions. I am trying them out and will let you know how they work ASAP. If successful I will write a summary and post to ccp4bb.
Best, Shengyang From: Conners, Becky [mailto:r.conn...@exeter.ac.uk] Sent: 10 July 2019 16:29 To: Shengyang Jin <jinshengy...@outlook.com> Subject: Re: MR for coiled coil structure Hi Shengyang, It is from a few years ago now, and there may be better methods now, but you could have a look at: EMBO J.<https://www.ncbi.nlm.nih.gov/pubmed/18497748> 2008 Jun 18;27(12):1779-89. doi: 10.1038/emboj.2008.101. Epub 2008 May 22. The Moraxella adhesin UspA1 binds to its human CEACAM1 receptor by a deformable trimeric coiled-coil. Conners R<https://www.ncbi.nlm.nih.gov/pubmed/?term=Conners%20R%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>1, Hill DJ<https://www.ncbi.nlm.nih.gov/pubmed/?term=Hill%20DJ%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>, Borodina E<https://www.ncbi.nlm.nih.gov/pubmed/?term=Borodina%20E%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>, Agnew C<https://www.ncbi.nlm.nih.gov/pubmed/?term=Agnew%20C%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>, Daniell SJ<https://www.ncbi.nlm.nih.gov/pubmed/?term=Daniell%20SJ%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>, Burton NM<https://www.ncbi.nlm.nih.gov/pubmed/?term=Burton%20NM%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>, Sessions RB<https://www.ncbi.nlm.nih.gov/pubmed/?term=Sessions%20RB%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>, Clarke AR<https://www.ncbi.nlm.nih.gov/pubmed/?term=Clarke%20AR%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>, Catto LE<https://www.ncbi.nlm.nih.gov/pubmed/?term=Catto%20LE%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>, Lammie D<https://www.ncbi.nlm.nih.gov/pubmed/?term=Lammie%20D%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>, Wess T<https://www.ncbi.nlm.nih.gov/pubmed/?term=Wess%20T%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>, Brady RL<https://www.ncbi.nlm.nih.gov/pubmed/?term=Brady%20RL%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>, Virji M<https://www.ncbi.nlm.nih.gov/pubmed/?term=Virji%20M%5BAuthor%5D&cauthor=true&cauthor_uid=18497748>. The structure was solved by a novel exploratory molecular replacement method using in excess of 20 different coiled-coil structures as search models in the program PHASER (McCoy et al., 2005). The coordinates of a single strand of the coiled coil of cortexellin (1D7M.pdb), which has no recognisable sequence identity to UspA1(527-665), was eventually successful despite being from a dimeric coiled coil. Solutions from PHASER had Z scores of 6 and 13 after rotation and translation respectively to find the first molecule in the asymmetric unit, and 5 and 9 for the second molecule. Initial phases were improved by mutation of the sequence to poly-Ala followed by "atoms update and refinement" mode of ARP/wARP (Perrakis et al., 2001). Symmetry operators were applied to the first monomer to create the two trimers. The structure was refined with iterative cycles of manual model-building using COOT (Emsley and Cowtan, 2004), restrained refinement with REFMAC5 (Murshudov et al., 1997) and density improvement with ARP/wARP as the correct sequence was gradually built into the improving electron density maps. Data collection and final refinement statistics are summarised in Table 1. The structure was a trimeric coiled coil, but the model we were most successful with was actually from a dimeric coiled coil and had no sequence identity to our protein. The "atom update and refinement" option in Arp/wARP was excellent at improving our maps. Good luck! Becky ---------------------------------------------------------------------------------------------------------------------- Dr Becky Conners Postdoctoral Research Associate University of Exeter 01392 727468 www.exeter.ac.uk<http://www.exeter.ac.uk> Office S03.07, Living Systems Institute, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK [Corporate email signature] This email and any attachment may contain information that is confidential, privileged, or subject to copyright, and which may be exempt from disclosure under applicable legislation. It is intended for the addressee only. If you received this message in error, please let me know and delete the email and any attachments immediately. The University will not accept responsibility for the accuracy/completeness of this email and its attachments. ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Shengyang Jin <jinshengy...@outlook.com<mailto:jinshengy...@outlook.com>> Sent: 10 July 2019 09:00 To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] MR for coiled coil structure Dear all, We recently acquired a data set (2.0 A, P222) for a coiled coil protein (according to Itasser, QUARK, Robetta, and Phaser). Matthews coefficient indicates 1 copy of protein per ASU. Sequence of the protein is quite novel with no apparent homolog in PDB. We tried to to MR with various models (ab initio or homology based) but with little success. We then tried to use AMPLE, but in ccp4 it always returned this error: __main__.py: error: unrecognized arguments: -use_arpwarp True (if we untick arpwarp and choose buccaneer instead, it returns -use_arpwarp False) Could anyone help? Thank you very much. Shengyang Jin Nanyang Technological University Singapore ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1&data=02%7C01%7CR.Conners%40exeter.ac.uk%7Cb0a055eecf9344e1208908d7050e2cd8%7C912a5d77fb984eeeaf321334d8f04a53%7C0%7C0%7C636983430750761781&sdata=f2UYtXHRgkVgN4F3h%2BGT6vJiqKq33U4edG7CWEf3TSw%3D&reserved=0> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
