Re: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)
Dear All After about 10 (!) years of (very) hard work we solved the structures of our dearest membrane transporter. Dataset at 2.9 And resolution, fairly anisotropic, experimental phasing, and many long nights with Coot and Buster to achieve model refinement. The experimental structure had a well defined ligand nicely coordinated but also a lipid embedded inside the binding cavity (a complete surprise but biologically relevant) and two detergent molecules well defined (experimental/crystallisation artefact). As our paper was accepted basically when CASP organisers were calling for targets I offered my baby to the computing Gods. However we only provided the sequence to CASP, no info regarding any ligand or lipid. Less than a month after, the CASP team contacted us and send us the best model. In fact it was 2 half models as the transporter is a pseudo dimer, with the N-lobe and C-lobe moving relative to each other during transport cycle, thus divided as two domains in CASP. The results were breathtaking. 0.7 And RSMD on one half, 0.6 on the other. And yes, group 427 was the superpower (did not know at the time that it was AlphaFold). We had long discussions with the CASP team, as -for us- this almost exact modelling was dream-like (or science fiction) and -at some point- we were even suspecting fraud, as our coordinates had travelled over the internet a few times around when interacting with colleagues. The organisers reassured us that we were not the only target that had been “nailed” so no reason to suspect any wrongdoing. To this day I am still baffled and I would be happy to hear from the community, maybe from some of the CASP participants. The target is T024, the “perfect" models are domain-split version (T024-D1 and T024-D2), as AlphaFold2 did not perform so well on the complete assembly. Deposited PDB is 6T1Z Cedric PS: I should also note that many other groups performed very well, much better than I would have dreamed, including on the full protein but just not as crazy-good. — Prof. Cedric Govaerts, Ph.D. Universite Libre de Bruxelles Campus Plaine. Phone :+32 2 650 53 77 Building BC, Room 1C4 203 Boulevard du Triomphe, Acces 2 1050 Brussels Belgium http://govaertslab.ulb.ac.be/ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] ADVX in remote acces is a nightmare
Hi all, I'm currently trying to collect data remotely (me sitting at home, crystals at Diamond Light Source). This involves accessing the beamline computer system via a NX machine system, thus opening a virtual desktop of the remote machine on my local computer (PC running W7) Now, I'm trying to look at the images via ADXV, which works fine on site, but close to impossible here. When I load the file in ADXV the main window is white, no images (all the menus are alive, it's reacting to the position of the pointer, giving resolution etc...). Sometimes (but not always!), I can see the image once I've dragged another window onto the ADVX window, this seems to refresh it and then I can work with it fine. But as of now, that happens very only rarely... thus I cannot see my images.. Is there a trick/workaround ? Thanks... (beamtime is running by..) Cedric
[ccp4bb] Crystal Quick low profile 96 wells plate definition for Mosquito
Hi all, I'm looking for the plate definition for the Greiner CrystalQuick low profile 2 wells plates, flat bottom, for the Mosquito. I could not find the plate definition for the 2 wells... eagerly looking for it... Thanks ! Cedric Cedric Govaerts, Ph.D. Universite Libre de Bruxelles Campus Plaine. Phone :+32 2 650 53 77 Building BC, Room 1C4 203 Boulevard du Triomphe, Acces 2 1050 Brussels Belgium,
[ccp4bb] Measuring protein concentration with 2mM ATP around
Dear all, We're doing some crystallization trials on a protein that requires 2mM ATP in the buffer and we are having trouble measuring reliably/reproducibly protein concentration. This is a real problem for optimization (last screen failed because of excessive protein concentration compared to the previous run). What we observe: -2mM ATP seems to prevent reliable estimation at 280nm with the nanodrop (albeit the Akta led detector seems to do OK at 280nm) due to the major contribution in the 200-300nm range. I guess blanking is difficult due to the relatively large contribution of ATP vs.protein at 280 -For some reason I cannot explain, Bradford measurment (using Pierce/Thermo dye) is also unreliable, comparable sample giving different values. I cannot see why ATP would do tha (buffer is 20mM Hepes, pH7.5, 150mM NaCl, 10 %Glycerol, 10% Ethylene glycol, 3mM MgCl2 and 2mM ATP). As this is for estimation of the protein concentration while concentrating before going into crystallization plates, the assay should be quick (minutes) and use little amount of material (say max 5µl per measure). An we're really interested in relative concentration (from one experiment to the next) rather than absolute value. I'm guessing as many of you have worked with ATPase etc, there must be a smart way to do this. Thanks for any input Cedric -- Cedric Govaerts, Ph.D. Universite Libre de Bruxelles Campus Plaine. Phone :+32 2 650 53 77 Building BC, Room 1C4 203 Boulevard du Triomphe, Acces 2 1050 Brussels Belgium
[ccp4bb] Postdoctoral position available : Crystallization of CFTR
Nanobody-aided crystallization and structure determination of CFTR Start date : march 1st 2013 The project: Cystic Fibrosis (CF or mucoviscidosis), is a fatal genetic disorder affecting one in 2500 newborn. It is caused by mutation in the CFTR, a chloride channel, leading to destabilization, degradation and malfunctioning of the protein. Obtaining the molecular structure of CFTR will represent a major breakthrough both for our understanding of the protein function /dysfunction but also to provide new avenues for therapeutic strategies. The project aims at obtaining the crystal structure of CFTR by combining cutting edge methodologies such as Lipidic Cubic Phase-based crystallography and nanobody stabilization of CFTR. Host laboratories: As a joint effort between 3 laboratories, the project is coordinated by Dr C. Govaerts at the Structure and Function of Membrane Biology Laboratory (SFMB,) affiliated with the Université Libre de Bruxelles and is located in Brussels, Belgium, the capital of Europe. A large part of the training and of the crystallization work will take place in the laboratory of Prof Martin Caffrey, Trinity College Dublin, Ireland, a pioneer in the LCP methodology. Finally, part of the work will be performed in the laboratory of Prof John Riordan, a discoverer of CFTR, located at University of North Carolina in Chappell Hill, USA. Profile: Candidates should have a PhD and have a background in biochemistry, protein expression and purification, preferably with membrane proteins. Experience with structural biology, specifically protein crystallization is a plus. The successful candidate must be creative, self-motivated, persistent, resourceful and be willing to travel between the different labs and enjoy working both independently and in a collaborative setting. Applications: Send a CV, a list of publications, a short overview of research activities and the name of two or more references to cedric.govae...@ulb.ac.be. Preselected applicants will be requested to travel to Brussels for a lecture and an interview.
