Re: [ccp4bb] Blob Game
...And the cryoprotectant was simply the Li/NH4SO4 mother liquor. Also maybe relevant: the crystals were pretty old, a year or so. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: Monday, February 23, 2015 12:21 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Blob Game Dear Crystallographers, I've got a strange blob hanging off an arginine--see attached. Any ideas? Nothing weird in the prep; cryst+prot conditions: NH4SO4, TRIS/HEPES, CaCl2, NaCl, Li2SO4. There are really no other side chains which could be close enough, and there's nothing in the anomalous LLG map. ??? JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] Blob Game
Ethanol? You weren't setting up drops with liquor on your breath, were you? I can't remember. JPK On 02/23/2015 12:20 PM, Keller, Jacob wrote: Dear Crystallographers, I've got a strange blob hanging off an arginine--see attached. Any ideas? Nothing weird in the prep; cryst+prot conditions: NH4SO4, TRIS/HEPES, CaCl2, NaCl, Li2SO4. There are really no other side chains which could be close enough, and there's nothing in the anomalous LLG map. ??? JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] Blob Game
a horrible water molecule would fit in there :-) On 23/02/15 19:42, Keller, Jacob wrote: A little more info: -Resolution is 1.75 Ang at edge of detector, but I/sig was still ~3; pushing integration to corners gives 1.66 Ang, but completeness is horrible. The home setup was at the limit (without 2 theta.) -Data are horribly twinned with fractions of 58/42%, spacegroup is P3212. Besides that, though, the data are really excellent. -Peak is 6.40 rmsd in Refmac-generated difference map. -Distance from blob-peak to Arg-N is 3.43 Ang, but the peak is obviously not spherical. JPK -Original Message- From: Keller, Jacob Sent: Monday, February 23, 2015 12:34 PM To: Keller, Jacob; CCP4BB@JISCMAIL.AC.UK Subject: RE: Blob Game ...And the cryoprotectant was simply the Li/NH4SO4 mother liquor. Also maybe relevant: the crystals were pretty old, a year or so. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: Monday, February 23, 2015 12:21 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Blob Game Dear Crystallographers, I've got a strange blob hanging off an arginine--see attached. Any ideas? Nothing weird in the prep; cryst+prot conditions: NH4SO4, TRIS/HEPES, CaCl2, NaCl, Li2SO4. There are really no other side chains which could be close enough, and there's nothing in the anomalous LLG map. ??? JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org *** -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Tel: +47 2284 0794
Re: [ccp4bb] Blob Game
A little more info: -Resolution is 1.75 Ang at edge of detector, but I/sig was still ~3; pushing integration to corners gives 1.66 Ang, but completeness is horrible. The home setup was at the limit (without 2 theta.) -Data are horribly twinned with fractions of 58/42%, spacegroup is P3212. Besides that, though, the data are really excellent. -Peak is 6.40 rmsd in Refmac-generated difference map. -Distance from blob-peak to Arg-N is 3.43 Ang, but the peak is obviously not spherical. JPK -Original Message- From: Keller, Jacob Sent: Monday, February 23, 2015 12:34 PM To: Keller, Jacob; CCP4BB@JISCMAIL.AC.UK Subject: RE: Blob Game ...And the cryoprotectant was simply the Li/NH4SO4 mother liquor. Also maybe relevant: the crystals were pretty old, a year or so. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: Monday, February 23, 2015 12:21 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Blob Game Dear Crystallographers, I've got a strange blob hanging off an arginine--see attached. Any ideas? Nothing weird in the prep; cryst+prot conditions: NH4SO4, TRIS/HEPES, CaCl2, NaCl, Li2SO4. There are really no other side chains which could be close enough, and there's nothing in the anomalous LLG map. ??? JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] Blob Game
Ethanol? You weren't setting up drops with liquor on your breath, were you? On 02/23/2015 12:20 PM, Keller, Jacob wrote: Dear Crystallographers, I've got a strange blob hanging off an arginine--see attached. Any ideas? Nothing weird in the prep; cryst+prot conditions: NH4SO4, TRIS/HEPES, CaCl2, NaCl, Li2SO4. There are really no other side chains which could be close enough, and there's nothing in the anomalous LLG map. ??? JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] Blob Game
Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob [kell...@janelia.hhmi.org] Sent: Monday, February 23, 2015 8:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Blob Game Ethanol? You weren't setting up drops with liquor on your breath, were you I can't remember. Ahah, so you did have some liquor. Good. Seriously, if there is something below the blob in the direction that you sent us and could be joined with the peak, couldn't an SO-4 fit in there somehow? It's too detailed for water at this resolution, isn't it? Boaz JPK On 02/23/2015 12:20 PM, Keller, Jacob wrote: Dear Crystallographers, I've got a strange blob hanging off an arginine--see attached. Any ideas? Nothing weird in the prep; cryst+prot conditions: NH4SO4, TRIS/HEPES, CaCl2, NaCl, Li2SO4. There are really no other side chains which could be close enough, and there's nothing in the anomalous LLG map. ??? JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] Blob Game
Just a little mother liquor. I am seeing what SO4 does in refinement right now. JPK -Original Message- From: Boaz Shaanan [mailto:bshaa...@bgu.ac.il] Sent: Monday, February 23, 2015 2:50 PM To: Keller, Jacob; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] Blob Game Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob [kell...@janelia.hhmi.org] Sent: Monday, February 23, 2015 8:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Blob Game Ethanol? You weren't setting up drops with liquor on your breath, were you I can't remember. Ahah, so you did have some liquor. Good. Seriously, if there is something below the blob in the direction that you sent us and could be joined with the peak, couldn't an SO-4 fit in there somehow? It's too detailed for water at this resolution, isn't it? Boaz JPK On 02/23/2015 12:20 PM, Keller, Jacob wrote: Dear Crystallographers, I've got a strange blob hanging off an arginine--see attached. Any ideas? Nothing weird in the prep; cryst+prot conditions: NH4SO4, TRIS/HEPES, CaCl2, NaCl, Li2SO4. There are really no other side chains which could be close enough, and there's nothing in the anomalous LLG map. ??? JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] Blob Game
on 23/2/15 6:57 PM, jens Preben Morth j.p.mo...@ncmm.uio.no wrote: a horrible water molecule would fit in there :-) On 23/02/15 19:42, Keller, Jacob wrote: A little more info: -Resolution is 1.75 Ang at edge of detector, but I/sig was still ~3; pushing integration to corners gives 1.66 Ang, but completeness is horrible. The home setup was at the limit (without 2 theta.) -Data are horribly twinned with fractions of 58/42%, spacegroup is P3212. Besides that, though, the data are really excellent. -Peak is 6.40 rmsd in Refmac-generated difference map. -Distance from blob-peak to Arg-N is 3.43 Ang, but the peak is obviously not spherical. JPK Jacob, Jens may be correct. Is it possible it is a water molecule mixed with an alternate conformation for the Arg side chain? Notice a little blob of positive density next to the nitrogen atom. Could the guanidino group be partly flipped? (Just a suggestion to consider it probably is not). If you cannot find a convincing model it is a good idea to add a UNK/UNX atom to indicate ambiguity. Good luck Oliver
