Re: [ccp4bb] How to reduce no. of overlaps
Jim Pflugrath wrote: In addition to reducing the beam divergence, you may wish to use a smaller beam size by using a smaller collimator or making the slits smaller. A smaller crystal can also help to spatially separate the Bragg spots as can moving the detector closer to the crystal. Yes, closer to the crystal. This is not intuitive, but arises since modern homelab beams are not parallel but are diverging from a focal point near the sample position. It is just something else you may wish to try. But the pattern is also diverging from a point at the sample? I'm guessing the focus point is somewhere between the crystal and the detector, so by moving the detector closer you are better approximating "focus on the detector" rather than "focus on the crystal"? With a home source one probably has room for a Huber goniometer with arcs, or better yet one of those goniometers that allows rotation up to 90* about a point at the crystal, so the crystal doesn't move out of the cold stream as you rotate. One can also cheat on the mosaicity during integration by fixing it at a small fraction of the true mosaicity. This is called "cutting off the wings" or more euphemistically "peak height sampling". The accuracy will suffer, but not as much as you might expect- probably because if spot profiles are pretty similar, the the height at peak is a good measure of peak volume. eab
Re: [ccp4bb] How to reduce no. of overlaps
In addition to all the excellent suggestions if you can you can also move your detector away from the center of the beam aka increase the detector size in one dimension. Not sure if you can do that at your home source though. By moving the center of the beam say to the lower 9/10 of the detector you will still have the beam position on the detector but you will also have increased the total area. Needless to say you will need to collect over a larger wedge to get a complete dataset. Regarding your overlap issues, it does help to spend some time thinking about the experiment before collecting the data, I would like to point toward the direction of Mosflm & strategy in conjunction with the Testgen option. Depending how badly your overlapped data is, you might want to run it through XDS and see how much it can rescue, assuming you used Mosflm or HKL2000. You can also give the Separation Close command a try in Mosflm with Postref Width and Profile Optimize (RTFM for more details). Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] How to reduce no. of overlaps
my experiences: C2 with a long axis parallel to the shortest crystal dimension, crystals were plates. Used prebent loops to fish the crystals. Personally I haven't tried to bend loops in mounted crystals as Frank does, but it sounds very useful. bar-shaped P321 crystals with hexagonal cross-sections, these naturally "fell" into loops in the "right" orientations. Too "right" usually, because you also don't want the long axis too parallel to the rotation axis and have a large blind cone compromising completeness, 10-20º from parallel is great, here a minikappa or the bendable loop mounts are very useful. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 7 Mar 2012, at 08:44, Frank von Delft wrote: > Yes... quite. Thanks! > > (Beam, rotation axis... how can you expect me to keep all these new-fangled > inventions apart?!) > > > > On 07/03/2012 07:33, VAN RAAIJ , MARK JOHAN wrote: >> typo correction, you'll want the long axis parallel to the rotation >> axis, not to the beam. >> Mark >> >> Quoting Frank von Delft: >> >>> You probably have to tilt your crystal, so that the long axis is >>> parallel to the beam. We do this routinely: cut a plastic pipette >>> tip to have a sharp point, then push the loop where it attaches to >>> the pin, to bend the crystal itself. >>> >>> You have to identify from your diffraction whether the long axis is >>> pointing through the face or the edge of the loop. As it's P6, >>> chances are it's through the face, because long-axis P6 tends to >>> make flat hexagons which lie flush with the face. So you have to >>> bend so the face of the loop upwards. >>> >>> You'll have to practice this first, though, so put up an empty loop. >>> Top tips: >>> * Don't breathe! You'll blow the cryostream away. >>> * Bend the loop towards (rather than away from) the rim edge of >>> the pin to which it's glued. >>> * Don't breathe! >>> * Practise practise practise. >>> >>> >>> Another thing: most in-house sources allow you to reduce divergence >>> of the beam. You lose intensity, but no matter, just expose longer. >>> That also improves overlap. >>> >>> Cheers >>> phx >>> >>> >>> >>> On 07/03/2012 04:56, Dipankar Manna wrote: Dear Crystallographers, I am working on a protein having SG P6, the cell parameters are a= 79, b= 79, c= 325. The crystals are forming in big size and with very good shape. It also diffracting very well in Home source facility both in terms of resolution and intensity. But the only problem is the number of overlaps. Its showing much more than the good spots. As a result the completeness is showing maximum up to 65% even after collecting 180 degrees. I am unable to get a complete data. I tried with reducing the oscillation angel to 0.3 degree/0.5 degree but it did not improve that much. Please give me some suggestions. Regards, Dipankar /Dipankar Manna/ */Aurigene Discovery Technologies Limited,/* /#39-40 KIADB Industrial Area, Electronic City, Phase II,/ /Hosur Road, Bangalore- 560 100, India/ /Cell: +91-9538631469 // | Office Ph : +91 80-66204422 (Extn: 398) | Email ID: dipanka...@aurigene.com/ This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com >> >> >> Mark J van Raaij >> Laboratorio M-4 >> Dpto de Estructura de Macromoléculas >> Centro Nacional de Biotecnología - CSIC >> c/Darwin 3, Campus Cantoblanco >> 28049 Madrid >> tel. 91 585 4616 >> email: mjvanra...@cnb.csic.es >>
Re: [ccp4bb] How to reduce no. of overlaps
Hi, Besides aligning the long axis with the rotation axis, which is the most important, there are a few more things that may help: 1) Try optimizing the freezing to reduce the mosaicity (if not ideal), or shoot at RT if possible. With higher mosaicity, the shape of the reflections are elongated in the reciprocal space, increasing the chance of overlapping. 2) Collect datesets in two runs, one with detector at far position to get more spacing between dots, another one at a closer position to get high res. Sometimes you may have to sacrifice some resolution for the completeness. 3) Shoot at a synchrotron with micro beam and a large detector. 4) Try integrating the images with both HKL2000 and mosflm. My past experience seems to suggest that mosflm is more tolerant to closely positioned reflections. Zhijie From: Dipankar Manna Sent: Tuesday, March 06, 2012 11:56 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to reduce no. of overlaps Dear Crystallographers, I am working on a protein having SG P6, the cell parameters are a= 79, b= 79, c= 325. The crystals are forming in big size and with very good shape. It also diffracting very well in Home source facility both in terms of resolution and intensity. But the only problem is the number of overlaps. Its showing much more than the good spots. As a result the completeness is showing maximum up to 65% even after collecting 180 degrees. I am unable to get a complete data. I tried with reducing the oscillation angel to 0.3 degree/0.5 degree but it did not improve that much. Please give me some suggestions. Regards, Dipankar Dipankar Manna Aurigene Discovery Technologies Limited, #39-40 KIADB Industrial Area, Electronic City, Phase II, Hosur Road, Bangalore- 560 100, India Cell: +91-9538631469 | Office Ph : +91 80-66204422 (Extn: 398) | Email ID: dipanka...@aurigene.com This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com
Re: [ccp4bb] How to reduce no. of overlaps
More accurately: "adjustable pin". Their *loops* are all adjustable (as opposed to Mitegen or MDL). Those pins are not very useful if your crystal is already mounted. And once you've bent that pin, it can't be stored again, because robots and tongs won't close around them. They do have the advantage of not sometimes bending back, the way bent loops do. On 07/03/2012 07:56, Zhijie Li wrote: Hampton sells an adjustable mounted loop for this purpose. http://hamptonresearch.com/product_detail.aspx?cid=24&sid=136&pid=385 http://hamptonresearch.com/product_detail.aspx?cid=24&sid=136&pid=385> *From:* Frank von Delft <mailto:frank.vonde...@sgc.ox.ac.uk> *Sent:* Wednesday, March 07, 2012 1:26 AM *To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> *Subject:* Re: [ccp4bb] How to reduce no. of overlaps You probably have to tilt your crystal, so that the long axis is parallel to the beam. We do this routinely: cut a plastic pipette tip to have a sharp point, then push the loop where it attaches to the pin, to bend the crystal itself. You have to identify from your diffraction whether the long axis is pointing through the face or the edge of the loop. As it's P6, chances are it's through the face, because long-axis P6 tends to make flat hexagons which lie flush with the face. So you have to bend so the face of the loop upwards. You'll have to practice this first, though, so put up an empty loop. Top tips: * Don't breathe! You'll blow the cryostream away. * Bend the loop towards (rather than away from) the rim edge of the pin to which it's glued. * Don't breathe! * Practise practise practise. Another thing: most in-house sources allow you to reduce divergence of the beam. You lose intensity, but no matter, just expose longer. That also improves overlap. Cheers phx On 07/03/2012 04:56, Dipankar Manna wrote: Dear Crystallographers, I am working on a protein having SG P6, the cell parameters are a= 79, b= 79, c= 325. The crystals are forming in big size and with very good shape. It also diffracting very well in Home source facility both in terms of resolution and intensity. But the only problem is the number of overlaps. Its showing much more than the good spots. As a result the completeness is showing maximum up to 65% even after collecting 180 degrees. I am unable to get a complete data. I tried with reducing the oscillation angel to 0.3 degree/0.5 degree but it did not improve that much. Please give me some suggestions. Regards, Dipankar /Dipankar Manna/ */Aurigene Discovery Technologies Limited,/* /#39-40 KIADB Industrial Area, Electronic City, Phase II,/ /Hosur Road, Bangalore- 560 100, India/ /Cell: +91-9538631469 // | Office Ph : +91 80-66204422 (Extn: 398) | Email ID: dipanka...@aurigene.com/ This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com
Re: [ccp4bb] How to reduce no. of overlaps
In addition to reducing the beam divergence, you may wish to use a smaller beam size by using a smaller collimator or making the slits smaller. A smaller crystal can also help to spatially separate the Bragg spots as can moving the detector closer to the crystal. Yes, closer to the crystal. This is not intuitive, but arises since modern homelab beams are not parallel but are diverging from a focal point near the sample position. It is just something else you may wish to try. How you flashcool your sample will also have a large effect on the spot sizes/shapes. Jim >. >Another thing: most in-house sources allow you to reduce divergence of the >beam. You lose intensity, >but no matter, just expose longer. That also >improves overlap. > >Cheers >phx
Re: [ccp4bb] How to reduce no. of overlaps
Hampton sells an adjustable mounted loop for this purpose. http://hamptonresearch.com/product_detail.aspx?cid=24&sid=136&pid=385 From: Frank von Delft Sent: Wednesday, March 07, 2012 1:26 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] How to reduce no. of overlaps You probably have to tilt your crystal, so that the long axis is parallel to the beam. We do this routinely: cut a plastic pipette tip to have a sharp point, then push the loop where it attaches to the pin, to bend the crystal itself. You have to identify from your diffraction whether the long axis is pointing through the face or the edge of the loop. As it's P6, chances are it's through the face, because long-axis P6 tends to make flat hexagons which lie flush with the face. So you have to bend so the face of the loop upwards. You'll have to practice this first, though, so put up an empty loop. Top tips: * Don't breathe! You'll blow the cryostream away. * Bend the loop towards (rather than away from) the rim edge of the pin to which it's glued. * Don't breathe! * Practise practise practise. Another thing: most in-house sources allow you to reduce divergence of the beam. You lose intensity, but no matter, just expose longer. That also improves overlap. Cheers phx On 07/03/2012 04:56, Dipankar Manna wrote: Dear Crystallographers, I am working on a protein having SG P6, the cell parameters are a= 79, b= 79, c= 325. The crystals are forming in big size and with very good shape. It also diffracting very well in Home source facility both in terms of resolution and intensity. But the only problem is the number of overlaps. Its showing much more than the good spots. As a result the completeness is showing maximum up to 65% even after collecting 180 degrees. I am unable to get a complete data. I tried with reducing the oscillation angel to 0.3 degree/0.5 degree but it did not improve that much. Please give me some suggestions. Regards, Dipankar Dipankar Manna Aurigene Discovery Technologies Limited, #39-40 KIADB Industrial Area, Electronic City, Phase II, Hosur Road, Bangalore- 560 100, India Cell: +91-9538631469 | Office Ph : +91 80-66204422 (Extn: 398) | Email ID: dipanka...@aurigene.com -- This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com
Re: [ccp4bb] How to reduce no. of overlaps
Yes... quite. Thanks! (Beam, rotation axis... how can you expect me to keep all these new-fangled inventions apart?!) On 07/03/2012 07:33, VAN RAAIJ , MARK JOHAN wrote: typo correction, you'll want the long axis parallel to the rotation axis, not to the beam. Mark Quoting Frank von Delft: You probably have to tilt your crystal, so that the long axis is parallel to the beam. We do this routinely: cut a plastic pipette tip to have a sharp point, then push the loop where it attaches to the pin, to bend the crystal itself. You have to identify from your diffraction whether the long axis is pointing through the face or the edge of the loop. As it's P6, chances are it's through the face, because long-axis P6 tends to make flat hexagons which lie flush with the face. So you have to bend so the face of the loop upwards. You'll have to practice this first, though, so put up an empty loop. Top tips: * Don't breathe! You'll blow the cryostream away. * Bend the loop towards (rather than away from) the rim edge of the pin to which it's glued. * Don't breathe! * Practise practise practise. Another thing: most in-house sources allow you to reduce divergence of the beam. You lose intensity, but no matter, just expose longer. That also improves overlap. Cheers phx On 07/03/2012 04:56, Dipankar Manna wrote: Dear Crystallographers, I am working on a protein having SG P6, the cell parameters are a= 79, b= 79, c= 325. The crystals are forming in big size and with very good shape. It also diffracting very well in Home source facility both in terms of resolution and intensity. But the only problem is the number of overlaps. Its showing much more than the good spots. As a result the completeness is showing maximum up to 65% even after collecting 180 degrees. I am unable to get a complete data. I tried with reducing the oscillation angel to 0.3 degree/0.5 degree but it did not improve that much. Please give me some suggestions. Regards, Dipankar /Dipankar Manna/ */Aurigene Discovery Technologies Limited,/* /#39-40 KIADB Industrial Area, Electronic City, Phase II,/ /Hosur Road, Bangalore- 560 100, India/ /Cell: +91-9538631469 // | Office Ph : +91 80-66204422 (Extn: 398) | Email ID: dipanka...@aurigene.com/ This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] How to reduce no. of overlaps
typo correction, you'll want the long axis parallel to the rotation axis, not to the beam. Mark Quoting Frank von Delft: You probably have to tilt your crystal, so that the long axis is parallel to the beam. We do this routinely: cut a plastic pipette tip to have a sharp point, then push the loop where it attaches to the pin, to bend the crystal itself. You have to identify from your diffraction whether the long axis is pointing through the face or the edge of the loop. As it's P6, chances are it's through the face, because long-axis P6 tends to make flat hexagons which lie flush with the face. So you have to bend so the face of the loop upwards. You'll have to practice this first, though, so put up an empty loop. Top tips: * Don't breathe! You'll blow the cryostream away. * Bend the loop towards (rather than away from) the rim edge of the pin to which it's glued. * Don't breathe! * Practise practise practise. Another thing: most in-house sources allow you to reduce divergence of the beam. You lose intensity, but no matter, just expose longer. That also improves overlap. Cheers phx On 07/03/2012 04:56, Dipankar Manna wrote: Dear Crystallographers, I am working on a protein having SG P6, the cell parameters are a= 79, b= 79, c= 325. The crystals are forming in big size and with very good shape. It also diffracting very well in Home source facility both in terms of resolution and intensity. But the only problem is the number of overlaps. Its showing much more than the good spots. As a result the completeness is showing maximum up to 65% even after collecting 180 degrees. I am unable to get a complete data. I tried with reducing the oscillation angel to 0.3 degree/0.5 degree but it did not improve that much. Please give me some suggestions. Regards, Dipankar /Dipankar Manna/ */Aurigene Discovery Technologies Limited,/* /#39-40 KIADB Industrial Area, Electronic City, Phase II,/ /Hosur Road, Bangalore- 560 100, India/ /Cell: +91-9538631469 // | Office Ph : +91 80-66204422 (Extn: 398) | Email ID: dipanka...@aurigene.com/ This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] How to reduce no. of overlaps
You probably have to tilt your crystal, so that the long axis is parallel to the beam. We do this routinely: cut a plastic pipette tip to have a sharp point, then push the loop where it attaches to the pin, to bend the crystal itself. You have to identify from your diffraction whether the long axis is pointing through the face or the edge of the loop. As it's P6, chances are it's through the face, because long-axis P6 tends to make flat hexagons which lie flush with the face. So you have to bend so the face of the loop upwards. You'll have to practice this first, though, so put up an empty loop. Top tips: * Don't breathe! You'll blow the cryostream away. * Bend the loop towards (rather than away from) the rim edge of the pin to which it's glued. * Don't breathe! * Practise practise practise. Another thing: most in-house sources allow you to reduce divergence of the beam. You lose intensity, but no matter, just expose longer. That also improves overlap. Cheers phx On 07/03/2012 04:56, Dipankar Manna wrote: Dear Crystallographers, I am working on a protein having SG P6, the cell parameters are a= 79, b= 79, c= 325. The crystals are forming in big size and with very good shape. It also diffracting very well in Home source facility both in terms of resolution and intensity. But the only problem is the number of overlaps. Its showing much more than the good spots. As a result the completeness is showing maximum up to 65% even after collecting 180 degrees. I am unable to get a complete data. I tried with reducing the oscillation angel to 0.3 degree/0.5 degree but it did not improve that much. Please give me some suggestions. Regards, Dipankar /Dipankar Manna/ */Aurigene Discovery Technologies Limited,/* /#39-40 KIADB Industrial Area, Electronic City, Phase II,/ /Hosur Road, Bangalore- 560 100, India/ /Cell: +91-9538631469 // | Office Ph : +91 80-66204422 (Extn: 398) | Email ID: dipanka...@aurigene.com/ This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com
[ccp4bb] How to reduce no. of overlaps
Dear Crystallographers, I am working on a protein having SG P6, the cell parameters are a= 79, b= 79, c= 325. The crystals are forming in big size and with very good shape. It also diffracting very well in Home source facility both in terms of resolution and intensity. But the only problem is the number of overlaps. Its showing much more than the good spots. As a result the completeness is showing maximum up to 65% even after collecting 180 degrees. I am unable to get a complete data. I tried with reducing the oscillation angel to 0.3 degree/0.5 degree but it did not improve that much. Please give me some suggestions. Regards, Dipankar Dipankar Manna Aurigene Discovery Technologies Limited, #39-40 KIADB Industrial Area, Electronic City, Phase II, Hosur Road, Bangalore- 560 100, India Cell: +91-9538631469 | Office Ph : +91 80-66204422 (Extn: 398) | Email ID: dipanka...@aurigene.com This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com
