[ccp4bb] To Trim or Not to To Trim

Hi All, I'm trying to crowdsource an opinion on how people deal with modelling side chains with poorly resolved electron or cryoEM density. My preference is to model the sidechain and allow the B-factors to go high in refinement to represent that the side chain is flexible. However, I'm aware tha

Re: [ccp4bb] Questionable Ligand Density - Part 2

Correction! I mean't 6BV0 et. al. Many apologies!!! Thanks Paul Brear for pointing this out. Rhys On Wed, 24 Jul 2019 at 21:36, Rhys Grinter wrote: > Hi All, > > Thanks for all the helpful comments and discussion surrounding my last > post. I've been doing a little m

[ccp4bb] Questionable Ligand Density - Part 2

ls. Best Wishes, Rhys -- Dr Rhys Grinter NHMRC Postdoctoral Researcher Monash University +61 (0)3 9902 9213 +61 (0)403 896 767 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin

[ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2

#x27;ve seen ligand bound structures with questionable density deposited in association with papers in well respected journals. Despite improvements to validation I feel that this problem is widespread. Best Regards, Rhys -- Dr Rhys Grinter NHMRC Postdoctoral Researcher Monash University +61 (0)3 990

[ccp4bb] PhD opportunity on Mycobacterial structural biology at Monash University, Melbourne

chris.green...@monash.edu. Please share with your colleagues and anyone interesting in starting a PhD in this exciting area. Best Wishes, Rhys -- Dr Rhys Grinter Sir Henry Wellcome Fellow Monash University +61 (0)3 9902 9213 +61 (0)403 896 767

[ccp4bb] Stable Refinement as Low(ish) resolution - Follow up

Hi All, Thanks for all the useful comments. Buster gave me quite stable refinement and nice maps, so I've been working with that so far. I'll work through the other suggestions however, and see what gives the best results. Cheers, Rhys -- Dr Rhys Grinter Sir Henry Wellcome Fel

[ccp4bb] Stable Refinement as Low(ish) resolution

community generally use when refining this kind of structure? Additionally Refmac doesn't seem to read the structure factors from the anisotropy server output file properly, giving vastly inflated R values and strange looking maps. Cheers, Rhys -- Dr Rhys Grinter Sir Henry Wellcome Fellow Monash

[ccp4bb] Twinning/Spacegroup Woes

is the best what to proceed with processing in this case? Cheers, Rhys -- Dr Rhys Grinter Sir Henry Wellcome Fellow Monash University +61 (0)3 9902 9213 +61 (0)403 896 767

[ccp4bb] Improving MR map contrast

rest of my protein? The solvent content of the crystal is around 55%. Cheers, -- Dr Rhys Grinter Sir Henry Wellcome Fellow Monash University +61 (0)3 9902 9213 +61 (0)403 896 767 -- Dr Rhys Grinter Sir Henry Wellcome Fellow Monash University +61 (0)3 9902 9213 +61 (0)403 896 767

Re: [ccp4bb] Post-translational modification of cystine

The large atom of the end of the modification looks wrong to me for BME. If it were BME it would just be an oxygen right? From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of RHYS GRINTER [r.grinte...@research.gla.ac.uk] Sent: 14 January 2015

Re: [ccp4bb] P31 or P32

erence when you do e.g. rigid body refinement. Best, Tim On 12/16/2014 10:39 AM, RHYS GRINTER wrote: > Hi All, > > This will no doubt show something of my ignorance with experimental phasing, > however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous > sign

[ccp4bb] P31 or P32

Hi All, This will no doubt show something of my ignorance with experimental phasing, however I'm currently working on solving a 3.8A SAD dataset with Pt anomalous signal. Both Shelx and Xtriage see reasonable anomalous signal to about 7A and seem to get statistics suggesting a solution when I

[ccp4bb] Tetrachloroplatinate coordination

Hi All, I was wondering if anyone knew off the top of their heads if there are any specific protein side chain residues which coordinate or react with Tetrachloroplatinate ions. I'm working on a low-res structure and was wondering if I could use the sites of platinum coordination as a starting

[ccp4bb] Heavy Atom Phasing

Hi All, I thought I might put a question to the community, with the hope of getting some tips of the best way to proceed with my heavy atom phasing problem. I'm working on solving the structure of an integral beta-barrel membrane protein of approximately 100 kDa. I've crystallised protein, growi

Re: [ccp4bb] Best compounds for heavy atom soaks

Hi All, A truly herculean response! Thanks everyone, I will process all of the information and come up with a strategy. Rhys

[ccp4bb] Best compounds for heavy atom soaks

Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see m

[ccp4bb] Bicelle Crystallisation

Hi All, I'm thinking of embarking on some crystallisation of a membrane protein in bicelles in the new year. In the methods I've read you simply take your protein in detergent and add the bicelle mxiture (Chapso+DMPC) to your protein allow the solution to equilibrate and set up your screens. Wh

[ccp4bb] tricky mr problem

Hi all, I have been attempting to find a MR solution for a low resolution data set (3.9A), with pretty poor merging stats of a 22 strand membrane beta barrel I'm working on. I've created a trimmed poly-alanine from a structure of 17% identity, that gives a solution with a Tfz of 14.3 with two

Re: [ccp4bb] Concentrating purified membrane protein

t it would minimize the volume to be concentrated (and thus the final detergent concentration). You could also try and intermediate cut off size concentrators you have available (20,30,50kDa etc) until you find biggest one that retain the sample. Good luck Rh

Re: [ccp4bb] Split Crystal Dataprocessing

13 16:09 To: RHYS GRINTER Subject: Re: [ccp4bb] Split Crystal Dataprocessing Dear Rhys I'm not sure if your group up there in Gla.ac.uk<http://Gla.ac.uk> uses "random" microseeding on a routine basis as soon as you get your first hits, but if you don't I would strongl

[ccp4bb] Split Crystal Dataprocessing

Hi All, I collected some data on the weekend on forked crystal, I collected data on this crystal at the base before the crystal split into two. The crystal didn't stand up well to the radiation damage so I shot a number of places along the crystal and got maybe 45 degrees of good data per positi

Re: [ccp4bb] Puzzling observation about size exclusion chromatography

Hi Zhen, I'm not sure that binding to a monoclonal antibody is good evidence that the protein is in a natively folded state. I would be suspicious of such a result as the protein could be improperly, which is causing it to interact with the column matrix. It could be useful to use some other te

Re: [ccp4bb] Calcium ions in enzymes

activity, and the presence of the Ca2+ ion in the structure. From: Eleanor Dodson [eleanor.dod...@york.ac.uk] Sent: 31 May 2013 12:49 To: RHYS GRINTER Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Calcium ions in enzymes I would think a Google search would

Re: [ccp4bb] Calcium ions in enzymes

My work with colicin M class bacteriocins shows that they require Ca2+ (or Mg or Mn) for catalysis: 1 Grinter, R., Roszak, A. W., Cogdell, R. J., Milner, J. J. and Walker, D. (2012) The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary R

Re: [ccp4bb] Membrane Protein Optimisation

lded crystals seemed biased for the formation of the gels. Thanks again for the help, I feel I might have a long raod to optimisation ahead of me. Rhys From: Jim Fairman [fairman@gmail.com] Sent: 09 May 2013 20:58 To: RHYS GRINTER Cc: ccp4bb Sub

[ccp4bb] Membrane Protein Optimisation

Hi All, A quick question if you've ever worked on membrane proteins, I'm trying to optimize crystals for bacterial integral outer membrane protein I'm working on. I'm getting some fairly modest rod like crystals in a 0.1M Tris pH 7.5, 30% PEG 600, 0.03M MgCl2 condition. However I get lots and

Re: [ccp4bb] salt or not?

What else in in the conditions? Calcium Sulphate/Phosphate is poorly soluble, so if there is any sulphate or phosphate in your condition I would be suspicious. The age of the plate is also a bad sign, as evaporation over an extended time can lead to salt crystals. Check the well solution for cry

Re: [ccp4bb] protein crystals or salt crystals

They look like other phosphate crystals that I've seen, but have to shoot them to tell. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols [nathaniel.ech...@gmail.com] Sent: 07 February 2013 22:30 To: CCP4BB@JISCMAIL.AC.UK Subject:

Re: [ccp4bb] side chain density

Hi Guys, I'm dealing with the same issue with a couple of structures at the moment. Matt you said that you like to leave the side chains in a plausible conformation and let refinement deal with the problem. Generally when I try this their backbone geometry gets all bent out of shape in refineme

[ccp4bb] Modelling Flexible Regions

if there was a consensus for when density is unmodelable, or if there are any tips for fitting residues into poor density. Thanks in advance, Rhys Grinter PhD Candidate University of Glasgow

Re: [ccp4bb] unidentified density

Is this density on an 2 fold axis? From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sudhir Kumar [sudhir.1...@gmail.com] Sent: 17 October 2012 12:07 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] unidentified density Dear all, I have been workin

[ccp4bb] Tricky MR problem

Thanks for your help everyone! It seems that the Balbes pipeline, followed by density modification in Phenix has done the trick Rhys

[ccp4bb] Tricky MR problem

o 2.7 angstroms with pretty nice stats. The space group is most likely C2221 with two molecules per ASU (giving around 58% solvent). Thanks, Rhys Grinter PhD Candidate University of Glasgow

Re: [ccp4bb] Improving microcrystals

Hi Samuel, I've has good success going from sphereulites to crystals using an additive screen (the 96 condition Hampton one is good) with the conditions giving the spherulites. Just watch for salt crystals as you'll be adding some compounds that might cause your Ca ions to form insoluble CaSO4.

[ccp4bb] Iron induced reduction of crystal

technique to create reducing conditions in protein crystallography, as the use of reducing agents isn't always practical. Cheers, Rhys Grinter PhD Candidate University of Glasgow

[ccp4bb] Molecular replacement of chimeric protein

to use all of this information in molecular replacement i.e. can you use two search models simultaneously in Phaser? Thanks again Rhys Grinter

[ccp4bb] Ferredoxin Containing Crystal Bleaching

assume this it due in some way to the radiation exposure but was wondering out curiosity whether someone had experienced this before and had a specific explanation for it. And if this will effect the integrity of the data. Cheers Rhys Grinter PhD Candidate University of Glasgow

Re: [ccp4bb] Expressed protein hinders cell lysis?

Hi, I often see no real change in change in solution appearance after sonication mediated lysis, with proteins which yield low amounts or no soluble protein in E. coli. I've had a look at the solution post lysis under the microscope and the cells are infact lysed, it's just the presence of high

[ccp4bb] Model submission

Dear All, I'm working out the finer details on a structural paper for submission to JBC. I'm having a slight problem with how to present my data. I've got a high resolution (1.46 A) truncated structure of the protein with the N-terminal 38aa removed. I've also got data from lower resolution (2.

[ccp4bb] Strange Density

NaCl 20mM Tris [pH 8.0] A colleague suggested that sulphate or phosphate could fit at these distances, but these ions have not been added at any stage of the crystallisation process. Could anyone give me some insight into what this density might represent? Thanks in advance, Rhys G