[ccp4bb] postdoc position in Vancouver - cryo-EM analysis of bioelectricity complexes
Dear colleagues, A postdoctoral position is available to work on the problem of coupling between different ion channels in electrically excitable tissues. https://www.vanpetegemlab.com/opportunities.html The project entails cryo-EM, electrophysiology and protein biophysics, and will make use of our state-of-the-art high-resolution EM facility ( https://cryoem.med.ubc.ca/), dedicated patch clamp and planar lipid bilayer electrophysiology setups, as well as all tools to express and purify proteins from bacterial and mammalian cells. We foster a diverse and inclusive lab environment and are looking for motivated lab members that share these values. Postdoctoral fellows receive the opportunity to join the Biochemistry Postdoctoral Scholar program at UBC. Joining the lab also means joining our local community of labs and clinicians interested in ion channels and their associated diseases. We are looking for motivated individuals with a recent PhD (obtained within the last 4 years) using either structural biology (crystallography, NMR, or cryo-EM) or electrophysiology. All applications can be sent to filip.vanpetegem'at'ubc.caPlease include an updated CV, names and contact details for 3 references, and describe your motivation to join the team. This is an excellent opportunity for structural biologists to learn complementary methods, or for electrophysiologists to get exposed to structural biology methods. Filip Van Petegem -- Filip Van Petegem, PhD Professor, Dept. of Biochemistry and Molecular Biology The University of British Columbia 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com Twitter: @FilipPetegem Bluesky: @filipvanpetegem website: https:///www.vanpetegemlab.com 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] postdoc position in protein design - industry/academia collaboration in Vancouver
*Postdoctoral opportunity in Protein Design and Structural Biology* A postdoctoral position is available in the laboratory of Prof. Filip Van Petegem at the University of British Columbia. The goal is to apply state of the art computational protein design methods to create novel protein molecules to enable structural cryo-EM characterization of therapeutic candidates and their target. The project is a collaboration with Amgen, one of the world’s leading biotechnology companies, and is supported through a MITACS scholarship. In recent years, antibody-based medicines have emerged as one of the most effective modalities to treat diverse illnesses from cancer and inflammation to metabolic and neurological disorders. The successful candidate will work with experts in computational design to create novel scaffold proteins and will gain hands-on experience in evaluating protein-protein interactions in an industrial setting. The individual we seek has a background in computational protein design and an interest in testing their designs in a laboratory setting. We are looking for a talented and highly motivated PhD graduate with a proven track record in biochemistry, bioengineering, computer sciences or a related discipline and a desire to push the envelope of biotherapeutic research. Experience in protein structure visualization (i.e. PyMOL) and design tools (i.e. Rosetta) is desired along with experience with protein purification and experimental characterization. This position offers a unique opportunity in early-stage drug discovery at the interface of academia and industry. Please email your statement of interest and CV to: Prof. Filip Van Petegem The University of British Columbia filip.vanpete...@gmail.com Dr. Christy A. Thomson Amgen British Columbia cthom...@amgen.com -- Filip Van Petegem, PhD Professor, Dept. of Biochemistry and Molecular Biology The University of British Columbia 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ Twitter: @FilipPetegem 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] postdoc position on cryo-EM of Ryanodine Receptors - Vancouver, Canada
Dear colleagues, A postdoctoral position is available to work on ion channel structural biology in the lab of Filip Van Petegemat the University of British Columbia (Vancouver, Canada). The project focuses on the regulation and pathophysiology of Ryanodine Receptors (RyRs) and their associated proteins. RyRs represent the largest ion channels currently known and are targets for hundreds of mutations that cause cardiac arrhythmias, myopathies, and malignant hyperthermia. The project combines cryo-EM and electrophysiology with various biophysical methods. For some aspects, X-ray crystallography may be required. Our lab has steady access to the in-house cryo-EM facility, which houses a Titan Krios with Falcon IV detector and a 200kV Glacios (Falcon III) for pre-screening grids, as well as microscopes for negative stain EM. Minimum requirements for applicants include a PhD in Biochemistry or a related discipline with extensive experience with one or more structural biology methods (NMR, X-ray crystallography, or cryo-EM). Direct experience with membrane proteins is an additional plus. The lab values diversity and inclusivity, and applicants from all backgrounds are encouraged to apply. This is an opportunity for people with a structural biology background to learn electrophysiological methods. Recent work from the lab includes an investigation of how Ryanodine Receptors are modulated by small molecules, disease mutations, and post-translational modification, using cryo-EM, X-ray crystallography, planar lipid bilayer electrophysiology, and enzyme kinetics. Recent work on this topic: Yang et al (2022) *PNAS* 119:e2120416119 Woll et al (2021) *Nature Comm.* 12:807 Ma et al (2020) *Nat Chem Biol*. 16,1246-1254 Haji-Ghassemi et al (2019) *Molecular Cell* 75,1-14 For more information on recent work, see http://crg.ubc.ca/VanPetegem/ The lab is housed within the Life Sciences Centre at UBC (lsi.ubc.ca), located on the Point Grey campus in Vancouver. Interested candidates should send a CV, a cover letter showing the motivation to apply, along with names and contact details for 3 references to filip.vanpete...@gmail.com. -- Filip Van Petegem, PhD Professor, Dept. of Biochemistry and Molecular Biology The University of British Columbia 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ Twitter: @FilipPetegem 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] postdoc position in cryo-EM and crystallography of ion channels in Vancouver, Canada
Dear colleagues, A postdoc position is available immediately at the University of British Columbia. The project combines X-ray crystallography, cryo-EM, and electrophysiology. Our lab has steady access to the in-house X-ray and cryo-EM facilities: - Microscopes: 300kV Titan Krios with Falcon IV detector and Selectris Energy filter. A 200kV Glacios (Falcon III detector) is available for prescreening grids - X-ray equipment in the lab includes Mosquito and Dragonfly robotics, and we have regular access to an in-house X-ray diffractor (Rigaku Micromax-007 HF with Saturn 744+ CCD detector) and various synchrotron beamlines Minimum requirements for applicants include a PhD in Biochemistry or a related discipline with extensive experience in either X-ray crystallography or cryo-EM, as well as protein expression and purification. Direct experience with membrane proteins is an additional plus. The lab values diversity and inclusivity, and applicants from all backgrounds are encouraged to apply. This is an opportunity for people with structural biology expertise to learn electrophysiological methods. Recent work from the lab includes an investigation of how Ryanodine Receptors are modulated by small molecules, disease mutations, and post-translational modification, using cryo-EM, X-ray crystallography, planar lipid bilayer electrophysiology, and enzyme kinetics. Woll et al (2021) *Nature Comm.* 12:807 Ma et al (2020) *Nat Chem Biol*. 16,1246-1254 Haji-Ghassemi et al (2019) *Molecular Cell* 75,1-14 For more information on recent work, see http://crg.ubc.ca/VanPetegem/ The lab is housed within the Life Sciences Centre at UBC (lsi.ubc.ca), located on the Point Grey campus in Vancouver. Interested candidates should send a CV, along with names and contact details for 3 references to *filip.vanpete...@gmail.com *. -- Filip Van Petegem, PhD Professor, Dept. of Biochemistry and Molecular Biology The University of British Columbia 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ Twitter: @FilipPetegem 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] postdoc positions at the University of British Columbia (Vancouver, Canada)
Dear colleagues, Two postdoctoral positions are available at The University of British Columbia in Vancouver, Canada: *Position 1:* A postdoctoral position is available immediately to work on ion channel structural biology in the lab of Filip Van Petegem, Department of Biochemistry and Molecular Biology. The project combines X-ray crystallography, cryo-EM, and electrophysiology. Our lab has steady access to the in-house X-ray and cryo-EM facilities: - Microscopes: 300kV Titan Krios with Falcon IV detector and Selectris Energy filter. A 200kV Glacios (Falcon III detector) is available for prescreening grids. - X-ray equipment in the lab includes Mosquito and Dragonfly robotics, and we have regular access to an in-house X-ray diffractor (Rigaku Micromax-007 HF with Saturn 744+ CCD detector) and various synchrotron beamlines Minimum requirement for applicants include a PhD in Biochemistry or a related discipline with extensive experience in either X-ray crystallography or cryo-EM, as well as protein expression and purification. Direct experience with membrane proteins is an additional plus. The lab values diversity and inclusivity, and applicants from all backgrounds are encouraged to apply. This is an opportunity for people with structural biology expertise to learn electrophysiological methods. Recent work from the lab includes an investigation of how Ryanodine Receptors are modulated by small molecules, disease mutations, and post-translational modification, using cryo-EM, X-ray crystallography, planar lipid bilayer electrophysiology, and enzyme kinetics. Woll et al (2021) *Nature Comm.* 12:807 Ma et al (2020) *Nat Chem Biol*. 16,1246-1254 Haji-Ghassemi et al (2019) *Molecular Cell* 75,1-14 For more information on recent work, see http://crg.ubc.ca/VanPetegem/ The lab is housed within the Life Sciences Centre at UBC (lsi.ubc.ca), located on the Point Grey campus in Vancouver. Interested candidates should send a CV, along with names and contact details for 3 references to *filip.vanpete...@gmail.com *. -- *Position 2:* A postdoctoral position is available to work on a joint project between the labs of Filip Van Petegem and Robert Molday, both in the Department of Biochemistry and Molecular Biology at the University of British Columbia. The project will involve Cryo-EM on neuronal membrane proteins and will make use of the excellent cryo-EM facility within the Life Sciences Institute. Microscopes available include a 300kV Titan Krios with Falcon IV detector and Selectris Energy filter. A 200kV Glacios (Falcon III) is available for prescreening grids. The structural biology will be complemented with various functional assays. The ideal candidate will have a PhD in Biochemistry or a related discipline, as well as extensive experience with either X-ray crystallography and/or Cryo-electron microscopy. Direct experience with membrane protein expression and mammalian cell cultures is an additional asset. The Molday and Van Petegem labs are housed within the Life Sciences Institute (https://lsi.ubc.ca/). For more information on the labs, see http://crg.ubc.ca/VanPetegem/ and https://biochem.ubc.ca/person/robert-molday/ Interested candidates should send a CV, along with names and contact details for 3 references to both mol...@mail.ubc.ca and filip.vanpete...@gmail.com. -- Filip Van Petegem, PhD Professor, Dept. of Biochemistry and Molecular Biology The University of British Columbia 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ Twitter: @FilipPetegem 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] Postdoctoral position in Vancouver, Canada - ion channel structural biology
Dear community, a postdoctoral position is available in the lab of Filip Van Petegem at the University of British Columbia. The project involves the structural and functional investigation of ion channels using cryo-EM and X-ray crystallography, supplemented with various functional assays. For a complete list of publications, see: https://pubmed.ncbi.nlm.nih.gov/?term=Van+Petegem+F=date The successful candidate should have ample experience with structural biology, either through X-ray crystallography and/or cryo-EM. Experience with the purification of membrane proteins is an additional asset. The project will make use of our in-house X-ray and cryo-EM facilities. The latter includes a 200kV Glacios and 300kV Titan Krios microscopes. Funding is available for 3 years, with an initial appointment of 1 year that is renewable. The lab is located in the Life Sciences Institute (https://lsi.ubc.ca/) at the UBC Point Grey Campus. UBC Interested individuals should send a CV and cover letter explaining their motivation and interest to the contact info given below. Please also include the contact details for 3 referees. The position will remain open until filled. Sincerely, Filip Van Petegem -- Filip Van Petegem, PhD Professor, Dept. of Biochemistry and Molecular Biology The University of British Columbia 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ Twitter: @FilipPetegem 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] postdoc position at the University of British Columbia, Vancouver.
Dear ccp4bb, A position is available for a highly motivated and skilled Post-doctoral Fellow (PDF) at the University of British Columbia in Vancouver, Canada. The successful candidate will lead cryo-EM experiments to understand potassium ion channel complex gating and drug regulation in the heart. The position will be held within the Life Sciences Centre and Departments of APT and Biochemistry and Molecular Biology at the University of British Columbia (UBC), under the co-supervision of Dr. David Fedida and Dr. Filip Van Petegem. The successful applicant must have a Ph.D. in structural biology. Excellent theoretical knowledge and practical skills in either X-ray crystallography or cryo-EM are required. Familiarity with the production and purification of membrane proteins is a major plus. The position forms part of a collaborative, multidisciplinary effort between the Fedida and Van Petegem laboratories. As such, excellent oral and written English communication skills, the proven ability to work and as part of a team, and strong individual research productivity are essential. Qualified Applicants should submit a cover letter including contact information for three professional references, and a curriculum vitae including a list of publications (*n.b.* do not include reprints). Application materials should be combined into a single PDF document and submitted via email to david.fed...@ubc.ca and filip.vanpete...@gmail.com The position is available immediately. The appointment will be at the rank of Postdoctoral Fellow with a salary commensurate with research experience, plus benefits (see https://hr.ubc.ca/benefits/eligibility-enrolment/eligibility-and-plan-cost/faculty-postdoctoral-fellows-employees-and ). *Equity and diversity are essential to academic excellence. An open and diverse community fosters the inclusion of voices that have been underrepresented or discouraged. We encourage applications from members of groups that have been marginalized on any grounds enumerated under the B.C. Human Rights Code, including sex, sexual orientation, gender identity or expression, racialization, disability, political belief, religion, marital or family status, age, and/or status as a First Nation, Metis, Inuit, or Indigenous person.* -- Filip Van Petegem, PhD Professor, Dept. of Biochemistry and Molecular Biology The University of British Columbia 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ Twitter: @FilipPetegem 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] postdoc in ion channel structural biology in Vancouver, British Columbia
The Department of Anesthesiology, Pharmacology and Therapeutics and Department of Biochemistry and Molecular Biology The University of British Columbia Vancouver, Canada Post-doctoral Fellow in Ion Channel Structural Biology A position is available for a highly motivated and skilled Post-doctoral Fellow (PDF) to lead structural biology efforts to understand potassium ion channel complex gating and drug regulation in the heart. The position will be held within the Life Sciences Centre and Departments of APT and Biochemistry and Molecular Biology at the University of British Columbia (UBC), under the co-supervision of Dr. David Fedida and Dr. Filip Van Petegem. The successful applicant must have a Ph.D. in structural biology. Excellent theoretical knowledge and practical skills in either X-ray crystallography or cryogenic electron microscopy are required. Likewise, familiarity with ion channels, patch clamp electrophysiology, and fluorescence spectroscopy of ion channels is beneficial. The position forms part of a collaborative, multidisciplinary effort between the Fedida and Van Petegem laboratories. As such, excellent oral and written English communication skills, the proven ability to work and as part of a team, and strong individual research productivity are essential. Qualified Applicants should submit a cover letter including contact information for three professional references, and a curriculum vitae including a list of publications (*n.b.* do not include reprints). Application materials should be combined into a single PDF document and submitted via email to david.fed...@ubc.ca and filip.vanpete...@gmail.com The closing date for applications is 15 April 2021. The position is available immediately. The appointment at the rank of Postdoctoral Fellow will initially be for one year with a salary commensurate with research experience, plus benefits (see https://hr.ubc.ca/benefits/eligibility-enrolment/eligibility-and-plan-cost/faculty-postdoctoral-fellows-employees-and ). *Equity and diversity are essential to academic excellence. An open and diverse community fosters the inclusion of voices that have been underrepresented or discouraged. We encourage applications from members of groups that have been marginalized on any grounds enumerated under the B.C. Human Rights Code, including sex, sexual orientation, gender identity or expression, racialization, disability, political belief, religion, marital or family status, age, and/or status as a First Nation, Metis, Inuit, or Indigenous person. * -- Filip Van Petegem, PhD Professor, Dept. of Biochemistry and Molecular Biology The University of British Columbia 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ Twitter: @FilipPetegem 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] postdoc position at the University of British Columbia
The Michael Smith Laboratories and Department of Biochemistry and Molecular Biology The University of British Columbia Vancouver, Canada Post-doctoral Fellow in Structural Biology A position is available for highly motivated and skilled Post-doctoral Fellow (PDF) to lead structural biology efforts to elucidate the molecular basis of complex carbohydrate utilization by the human gut microbiota. The position will be held within the Michael Smith Laboratories (MSL) and Department of Biochemistry and Molecular Biology at the University of British Columbia (UBC), under the co-supervision of Prof. Harry Brumer and Prof. Filip Van Petegem. The successful applicant must have a Ph.D. in structural biology. Excellent theoretical knowledge and practical skills in X-ray crystallography are required. Experience with cryogenic electron microscopy is advantageous. Likewise, familiarity with carbohydrates, carbohydrate-active enzymes, and carbohydrate-binding proteins is beneficial. The position forms part of a collaborative, multidisciplinary effort between the Brumer and Van Petegem laboratories. As such, excellent oral and written English communication skills, the proven ability to work and as part of a team, and strong individual research productivity are essential. Qualified Applicants should submit a cover letter including contact information for three professional references, and a curriculum vitae including a list of publications (*n.b.* do not include reprints). Application materials should be combined into a single PDF document and submitted via email to bru...@msl.ubc.ca and filip.vanpete...@gmail.com The closing date for applications is 15 March 2021. The position is available immediately. The appointment at the rank of Postdoctoral Fellow will initially be for one year with a salary commensurate with research experience, plus benefits (see https://hr.ubc.ca/benefits/eligibility-enrolment/eligibility-and-plan-cost/faculty-postdoctoral-fellows-employees-and ). *Equity and diversity are essential to academic excellence. An open and diverse community fosters the inclusion of voices that have been underrepresented or discouraged. We encourage applications from members of groups that have been marginalized on any grounds enumerated under the B.C. Human Rights Code, including sex, sexual orientation, gender identity or expression, racialization, disability, political belief, religion, marital or family status, age, and/or status as a First Nation, Metis, Inuit, or Indigenous person. * -- Filip Van Petegem, PhD Professor, Dept. of Biochemistry and Molecular Biology The University of British Columbia 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ Twitter: @FilipPetegem 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] wincoot recovering sessions
Hello all, using some large maps, my Wincoot likes to crash every now and then. I'd like to be able to recover the various maps and models I have open simultaneously, without having to read in everything again from scratch to get the same parameters. Has anybody had any luck using the 'recover session' in Wincoot? I just get the 'there's nothing to recover' message. Other than the obvious comment of switching to Linux or Mac, I'd be curious to see if anybody has been able to restore sessions in Wincoot, or has found another workaround for having to re-read and re-adjust everything. best regards, Filip Van Petegem -- Filip Van Petegem, PhD Professor, Dept. of Biochemistry and Molecular Biology The University of British Columbia 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] postdoc position in Vancouver - University of British Columbia
*Postdoctoral position - Membrane protein Biochemist * We are looking for a protein biochemist to join our team. We are investigating the structure and function of ion channels, membrane proteins that generate electrical signals in excitable cells. They are targets for disease mutations linked to cardiac arrhythmia, epilepsy, myopathies, and much more. We combine high-resolution techniques (X-ray crystallography, cryo-EM) with functional methods (calorimetry, electrophysiology) to obtain a complete understanding of their function. Our institute is equipped with state-of-the-art equipment for structural biology, including a new Titan Krios microscope with FalconIII detector and Volta phase plate, Vitrobots and screening microscopes (Tecnai Spirit and F20), 2 Rigaku 007 instruments for X-ray diffraction, and various robotics for setting up and visualizing crystallization screens. Your profile: * A recent PhD (obtained within the last 4 years) * Experience with structural biology (NMR, X-ray Crystallography or cryo-EM). However, applicants with excellent skills in membrane protein expression and purification, especially in mammalian or insect cell lines, are also encouraged to apply. For more info about our research program, please visit our website: http://crg.ubc.ca/VanPetegem/ The position is available immediately and remains open until filled. Interested applicants can send their CV, a cover letter, and names + contact details of 3 references to: filip.vanpete...@gmail.com -- Filip Van Petegem, PhD Professor, Dept. of Biochemistry and Molecular Biology The University of British Columbia 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] How to compare B-factors between structures?
Good point; I've tested this (n=1) in the past with a high-resolution dataset (synchrotron data) and low-resolution dataset (in-house) of crystals of the same protein grown in the same drop. Same space group, same unit cell. B-factors for the low-resolution dataset were higher. After dividing every individual B-factor by the average B-factor of each, the normalized-B-factor-versus-residue plot was identical for both structures. Adding or subtracting a constant value didn't do that. As I pointed out, this is only n=1, but comparing the high-and low-resolution structures of the same condition should give the answer as to which B-factor normalization is the most appropriate. Filip Van Petegem On Mon, Mar 4, 2013 at 11:16 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: You only entertain addition+subtraction--why not use multiplication/division to normalize the b-factors? JPK On Mon, Mar 4, 2013 at 2:04 PM, James Holton jmhol...@lbl.gov wrote: Formally, the best way to compare B factors in two structures with different average B is to add a constant to all the B factors in the low-B structure until the average B factor is the same in both structures. Then you can compare apples to apples as it were. The extra B being added is equivalent to blurring the more well-ordered map to make it match the less-ordered one. Subtracting a B factor from the less-ordered structure is sharpening, and the reason why you shouldn't do that here is because you'd be assuming that a sharpened map has just as much structural information as the better diffracting crystal, and that's obviously no true (not as many spots). In reality, your comparison will always be limited by the worst-resolution data you have. Another reason to add rather than subtract a B factor is because B factors are not really linear with anything sensible. Yes, B=50 is more disordered than B=25, but is it twice as disordered? That depends on what you mean by disorder, but no matter how you look at it, the answer is generally no. One way to define the degree of disorder is the volume swept out by the atom's nucleus as it vibrates (or otherwise varies from cell to cell). This is NOT proportional to the B-factor, but rather the 3/2 power of the B factor. Yes, 3/2 power. The value of B, is proportional to the SQUARE of the width of the probability distribution of the nucleus, so to get the volume of space swept out by it you have to take the square root to get something proportional the the width and then you take the 3rd power to get something proportional to the volume. An then, of course, if you want to talk about the electron cloud (which is what x-rays see) and not the nuclear position (which you can only see if you are a neutron person), then you have to add a B factor of about 8 to every atom to account for the intrinsic width of the electron cloud. Formally, the B factor is convoluted with the intrinsic atomic form factor, but a native B factor of 8 is pretty close for most atoms. For those of you who are interested in something more exact than proportional the equation for the nuclear probability distribution generated by a given B factor is: kernel_B(r) = (4*pi/B)^1.5*exp(-4*pi^2/B*r^**2) where r is the distance from the average position (aka the x-y-z coordinates in the PDB file). Note that the width of this distribution of atomic positions is not really an error bar, it is a range. There's a difference between an atom actually being located in a variety of places vs not knowing the centroid of all these locations. Remember, you're averaging over trillions of unit cells. If you collect a different dataset from a similar crystal and re-refine the structure the final x-y-z coordinate assigned to the atom will not change all that much. The full-width at half-maximum (FWHM) of this kernel_B distribution is: fwhm = 0.1325*sqrt(B) and the probability of finding the nucleus within this radius is actually only about 29%. The radius that contains the nucleus half the time is about 1.3 times wider, or: r_half = 0.1731*sqrt(B) That is, for B=25, the atomic nucleus is within 0.87 A of its average position 50% of the time (a volume of 2.7 A^3). Whereas for B=50, it is within 1.22 A 50% of the time (7.7 A^3). Note that although B=50 is twice as big as B=25, the half-occupancy radius 0.87 A is not half as big as 1.22 A, nor are the volumes 2.7 and 7.7 A^3 related by a factor of two. Why is this important for comparing two structures? Since the B factor is non-linear with disorder, it is important to have a common reference point when comparing them. If the low-B structure has two atoms with B=10 and B=15 with average overall B=12, that might seem to be significant (almost a factor of two in the half-occupancy volume) but if the other structure has an average B factor of 80, then suddenly 78 vs 83 doesn't seem all that different (only a 10% change
Re: [ccp4bb] Puzzling electron density upon heavy metal soaking
Hello Joern, do you have an image of the anomalous difference map as well? Do you see the same 7 peaks there, or only the central blob? Filip On Thu, Nov 1, 2012 at 11:45 AM, Joern Krausze jk...@helmholtz-hzi.dewrote: Dear all, I have two isomorphous crystals of the same protein. One crystal, let's call it 'derivative', was soaked with 10 mM CoCl2 whereas the other, let's call it 'original', was not. Both crystals were otherwise grown under identical conditions (see below) and treated the same. In the derivative, I see some puzzling electron density which I do not see in the original (find two pictures under the links below; the waters in the figures are just put in for your convenience, they were no part of the refinement). The density forms a planar hexagon of spheres with another sphere in the exact center. The distance from corner to corner is about 3.0 A but varies. The distance from one corner to the center is about 3.1 A. The hexagon is almost symmetrical as the angles enclosed by two edges are about 120° each. This density shows up at two positions, once located between two aspartate residues and once between an aspartate and a tyrosine residue. These are, however, no special postions. It seems obvious that this density is caused by the presence of Co(II) since it only shows up in the derivative and also coincides with peaks in the difference fourier map. I am unable to interpret it in a way that makes sense. Could anyone of you help me figure out what to build in there? Crystallization condition for both original and derivative was 200 mM Tris pH 8.8, 200 mM NaCl, 21% PEG 6000, 20% glycerol. The high-resolution limit of the dataset is 2.0 A. Data were collected at lambda=0.981 A. Anomalous data were collected at Co peak wavelength (weak SigAno but good enough for difference fourier map). links: http://imageshack.us/a/img89/**9376/unknowndensity1q.pnghttp://imageshack.us/a/img89/9376/unknowndensity1q.png http://imageshack.us/a/img696/**7135/unknowndensity2.pnghttp://imageshack.us/a/img696/7135/unknowndensity2.png Thank you in advance! Joern Address: Joern Krausze Molecular Structural Biology Helmholtz Centre for Infection Research Inhoffenstrasse 7 38124 Braunschweig Germany Email: joern.krau...@helmholtz-hzi.de Phone: +49 (0)531 6181 7023 (office) +49 (0)531 6181 7020 (lab) __**__ Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124 Braunschweig | www.helmholtz-hzi.de Vorsitzende des Aufsichtsrates: MinDir’in Bärbel Brumme-Bothe, Bundesministerium für Bildung und Forschung Stellvertreter: Rüdiger Eichel, Abteilungsleiter Niedersächsisches Ministerium für Wissenschaft und Kultur Geschäftsführung: Prof. Dr. Dirk Heinz; Ulf Richter, MBA Gesellschaft mit beschränkter Haftung (GmbH) Sitz der Gesellschaft: Braunschweig Handelsregister: Amtsgericht Braunschweig, HRB 477 -- Filip Van Petegem, PhD Associate Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Rigid body refinement with phenix using EM maps.
Hello Wojtek, a more straightforward approach is to use the rigid body refinement implemented in Situs. http://situs.biomachina.org/fguide.html After placing your pdb into the map via the colores program, you can refine the positions of individual portions using collage, by splitting your pdb into separate pdb files for each rigid body. No need to do any conversion. All you need is the pdb and the EM map downloaded from the EMDB. All are very simple to run and the documentation is very good. Regards, Filip -- Filip Van Petegem, PhD Associate Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ On Fri, Jul 13, 2012 at 8:28 AM, Wojtek Potrzebowski wpotr...@genesilico.pl wrote: Dear all, I want to refine a model of the complex that consists of rigid subunits using an electron microscopy electron density map. I've tried to use phenix for this purpose. Thus I have to back-FT the electron density map to a mtz reflection file. I've experienced problem using sfall for this purpose, but cinvfft has done a job (I can read a resulting file with mtzdmp). However phenix doesn't read this file properly (data labels box is empty). Can anyone help me on this, please? I am looking forward to hearing from you, Wojtek
[ccp4bb] estimate of effective concentration
Dear crystallographers, I have a question concerning effective concentration. Say you have a crystal structure whereby two loops, each part of a different domain but within the same molecule happen to be juxtaposed and can form an interaction. The loops have some degree of flexibility, but are ordered when interacting. The domains on which they are attached have a rigid configuration due to the remainder of the structure. The interaction is potentially very weak and mainly driven by the fact that the effective concentration is extremely high. The question: how can one obtain a rough estimate of the effective concentration of these two juxtaposed loops? The simple straightforward answer would be to just divide number (1 each) by volume (some box drawn around the loops), and convert this to molar. That's easy. However, this is over-simplified and really an underestimate of 'effective' concentration, because these loops cannot rotate freely when attached to the domains. Hence, there are constraints that allow them to interact more readily compared to the isolated loops within the same box. So I'm looking for a model that also takes limited conformational freedom into account. If anybody has any pointers to some reference text or paper that has performed such an analysis, I would be very interested. Regards, Filip -- Filip Van Petegem, PhD Associate Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
[ccp4bb] Movements of domains
Dear crystallographers, I have a general question concerning the comparison of different structures. Suppose you have a crystal structure containing a few domains. You also have another structure of the same, but in a different condition (with a bound ligand, a mutation, or simply a different crystallization condition,...). After careful superpositions, you notice that one of the domains has shifted over a particular distance compared to the other domains, say 1-1.5 Angstrom. This is a shift of the entire domain. Now how can you know that this is a 'significant' change? Say the overall resolution of the structures is lower than the observed distance (2.5A for example). Now saying that a 1.5 Angstrom movement of an entire domain is not relevant at this resolution would seem wrong: we're not talking about some electron density protruding a bit more in one structure versus another, but all of the density has moved in a concerted fashion. So this would seem 'real', and not due to noise. I'm not talking about the fact that this movement was artificially caused by crystal packing or something similar. Just for whatever the reason (whether packing, pH, ligand binding, ...), you simply observe the movement. So the question is: how you can state that a particular movement was 'significantly large' compared to the resolution limit? In particular, what is the theoretical framework that allows you to state that some movement is signifcant? This type of question of course also applies to other methods such as cryo-EM. Is a 7A movement of an entire domain 'significant' in a 10A map? If it is, how do we quantify the significance? If anybody has a great reference or just an individual opinion, I'd like to hear about it. Regards, Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Movements of domains
Hello Jacob, that's correct, I'm only looking at the mathematical significance, not the biological one. I follow the same reasoning - it is highly improbably for all atoms to be skewed in the same direction. In a case I'm currently looking at, I'm particularly dealing with cryo-EM data, not X-ray structures, but with the same underlying principles: what are the odds that all pixels of the map move together in the same direction? As mentioned for X-ray structures, a Luzzati analysis may give information about the positional errors, but there should be an increased resolution when comparing domain movements, because it's unlikely for all atoms to have an error in the same direction. Filip On Mon, Nov 21, 2011 at 2:16 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Just to clarify: I think the question is about the mathematical sense of significance, and not the functional or physiological significance, right? If I understand the question correctly, wouldn't the reasoning be that admittedly each atom in the model has a certain positional error, but all together, it would be very unlikely for all atoms to be skewed in the same direction? Jacob On Mon, Nov 21, 2011 at 4:04 PM, Filip Van Petegem filip.vanpete...@gmail.com wrote: Dear crystallographers, I have a general question concerning the comparison of different structures. Suppose you have a crystal structure containing a few domains. You also have another structure of the same, but in a different condition (with a bound ligand, a mutation, or simply a different crystallization condition,...). After careful superpositions, you notice that one of the domains has shifted over a particular distance compared to the other domains, say 1-1.5 Angstrom. This is a shift of the entire domain. Now how can you know that this is a 'significant' change? Say the overall resolution of the structures is lower than the observed distance (2.5A for example). Now saying that a 1.5 Angstrom movement of an entire domain is not relevant at this resolution would seem wrong: we're not talking about some electron density protruding a bit more in one structure versus another, but all of the density has moved in a concerted fashion. So this would seem 'real', and not due to noise. I'm not talking about the fact that this movement was artificially caused by crystal packing or something similar. Just for whatever the reason (whether packing, pH, ligand binding, ...), you simply observe the movement. So the question is: how you can state that a particular movement was 'significantly large' compared to the resolution limit? In particular, what is the theoretical framework that allows you to state that some movement is signifcant? This type of question of course also applies to other methods such as cryo-EM. Is a 7A movement of an entire domain 'significant' in a 10A map? If it is, how do we quantify the significance? If anybody has a great reference or just an individual opinion, I'd like to hear about it. Regards, Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] cryo protection
Hello Leonard, one thing to test is whether transferring your crystals to a drop containing simply well solution also causes cracking. If yes, then the possibility exists that the absence of protein in solution is causing the trouble. In that case, you can transfer the crystals to oil: you'll be transferring the solution (with protein) in which the crystal grew as well, and slowly remove it without adding anything 'different'. However, if your crystals crack simply because they are mechanically fragile, then the oil may actually be worse. Filip On Wed, Oct 26, 2011 at 9:46 AM, Leonard Thomas lmtho...@ou.edu wrote: Hi All, I have run into a very sensitive crystals system when it comes to cryo protecting them. I have run through the usual suspects and trays are going to be setup with a cryo protectant as part of crystallization cocktail. The one problem that seems to be occurring is that the crystals crack as soon as they are transfered out of the original drop. I am running out of ideas and really would love some new ones. Thanks in advance. Len Leonard Thomas Ph.D. Macromolecular Crystallography Laboratory Manager University of Oklahoma Department of Chemistry and Biochemistry Stephenson Life Sciences Research Center 101 Stephenson Parkway Norman, OK 73019-5251 lmtho...@ou.edu http://barlywine.chem.ou.edu Office: (405)325-1126 Lab: (405)325-7571 -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong
Hello, James Holton can probably tell more about this, but it is possible to create a library of potential coiled coil structures with differences in number of residues, superhelical radius, and residues per superhelical turn. A library of 300 theoretical coiled coils was generated and, in conjunction with EPMR, was used successfully to solve the structure of a KCNQ tetrameric coiled coil. See: Howard et al (2007) Neuron 53(5),663-675. And I second Sergei Strelkov's comment: what should be an expected tetramer could show up as a trimer, etc... so you may want to check via ultracentrifugation that you have the expected stoichiometry. Regards, Filip On Mon, Oct 17, 2011 at 10:09 AM, Napoleão Valadares n...@ifsc.usp.brwrote: Hi there! I got crystals from some synthetic peptides I bought, they are 30 residues long and are supposed to form a coiled coil. I collected various data sets (home source, Brookhaven and Diamond), including some at the resolution of 1.65 A, for which the space group appears to be C222 or C2221. The unit cell is small, 22.67, 88.06, 26.13, and the Matheus Coefficient indicates that's there's only one helix in the asymmetric unit and a 25% solvent content. I have tried A LOT of Molecular Replacement using Phaser and Phenix AutoMR. I'm using a 80% identity coiled coil helix as search model. The programs give me solutions with reasonable maps, but it is never possible to refine to achieve Rvalues below 0.40. Additionally, maps from different solutions look reasonable, so I'm thinking these are all bias. I have 5 other synthetic 30 residues peptides (that crystallize in different space groups and diffract to lower resolutions), including a SelenoMethionine (SM) derivative (but it does not have enough anomalous signal, ASU is too big, it is possible that the SM are disordered). I'm stuck on this since March. Regarding the search model, I already tried trimming some or all side chains and removing 2, 3 or 5 residues on each/both sides. I also tried other search models. Maybe some magic combination of parameters on Phaser or other programs can help me. What is your advice regarding how to proceed with MR? Is there some program, procedure, parameter, pray or human sacrifice that could help me? Thank you. Regards, Napo -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Protein elution in Size Exclusion
worst-case scenario for crystallization purposes: 9. Your protein runs as a mixture of monomers, dimers, trimers, whatever-mers... Filip On Sun, Aug 28, 2011 at 7:24 AM, David Briggs drdavidcbri...@gmail.comwrote: Following on from Roger's fine suggestions: 8. Your column is knackered. Can you see fine lines or cracks in the column? Good packing is v.important for SEC columns. HTH, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 28 August 2011 10:25, Allan Pang a.p...@qmul.ac.uk wrote: Hi there everyone, What does it mean when you have proteins eluting in almost the whole column volume of S200? I ran a gel with fractions from 8ml to 20ml and saw band for my protein all throughout. Judging peaks on chromatogram is not useful as it doesn't have any aromatic rings. Cheers, Allan -- Allan Pang PhD Student G35 Joseph Priestley Building Queen Mary University of London London E1 4NS Phone number: 02078828480 -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Another paper structure retracted
Just another example of where it would have been good for the reviewers to get access to the data during the review process... and where at least one of the reviewers *should* be a protein crystallographer... Filip Van Petegem On Wed, Aug 10, 2011 at 2:01 PM, David Schuller dj...@cornell.edu wrote: Time to fuel up the gossip engines for the approaching weekend: http://www.sciencedirect.com/science/article/pii/S096921260800186X RETRACTED: Structure of the Parathyroid Hormone Receptor C Terminus Bound to the G-Protein Dimer Gβ1γ2 Structure, Volume 16, Issue 7http://www.sciencedirect.com/science?_ob=PublicationURL_tockey=%23TOC%236269%232008%23999839992%23693753%23FLA%23_cdi=6269_pubType=Jview=c_auth=y_acct=C22719_version=1_urlVersion=0_userid=492137md5=9dc4b8953d3fa243dc98e395b6ac590d, 9 July 2008, Pages 1086-1094 Structure 2QNS withdrawn. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.
Dear Arnon, the Bradford method is not recommended for accurate measurements. The readings are strongly dependent on the amino acid composition. A much better method is using the absorption at 280nm under denaturing conditions (6M Guanidine), and using calculated extinction coefficients based on the composition of mostly Tyrosine and Tryptophan residues (+ disulfide bonds). This method is also old (Edelhoch, 1967), but very reliable. One thing about the nanodrop: smaller volume = more evaporation. On the demo we've had, I was so unimpressed with the precision (25% variability between two consecutive measurement) that we didn't consider this instrument at all. So unless you just want a 'rough' estimate, I wouldn't recommend it at all. But most respectable spectrophotometers will take cuvettes with 50ul volumes - a big step up from 1ml volumes... Filip Van Petegem On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie la...@uic.edu wrote: Dear fellow crystallographers - a question about spectrophotometers for protein concentration determination. We are so last millennium - using Bradford reagent/ 1 ml cuvette for protein conc. determination. We have been considering buying a Nanodrop machine (small volume, no dilution needed, fast, easy). However, while testing our samples using a colleague's machine, we have gotten readings up to 100% different to our Bradford assay (all fully purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. So while it is fun/easy to use the Nanodrop, I am not sure how reliable are the measurements (your thoughts?). So QUESTION 1: What are people's experience regarding the correlation between Nanodrop and Bradford? While researching the Nanodrop machine, I heard about the Implen NanoPhotmeter Pearl. So Question 2: Is the Pearl better/worse/same as the Nanodrop for our purpose? Thank you for helping us to advance to the next millennium, even if it is nearly a dozen years late. Arnon -- *** Arnon Lavie, Professor Dept. of Biochemistry and Molecular Genetics University of Illinois at Chicago 900 S. Ashland Ave. Molecular Biology Research Building, Room 1108 (M/C 669) Chicago, IL 60607 U.S.A. Tel:(312) 355-5029 Fax:(312) 355-4535 E-mail: la...@uic.edu http://www.uic.edu/labs/lavie/ *** -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.
25% is not acceptable for ITC or CD experiments though... I was just sharing our bad experience with a demo nanodrop we had. Even if evaporation is not an issue, one has to take pipetting errors into account when dealing with small volumes. The relative error on 1-2ul is a lot bigger than on 50ul. Unless you want to pre-mix 50ul and use a small quantity of that, which defeats the purpose of miniaturization... It all depends on your applications and sample availability, but if you want a very accurate measurement, miniaturized volumes just won't get you the same accuracy. Cuvettes will give a better accuracy provided you clean them properly. Just some water or EtOH is *not* enough... Filip Van Petegem On Thu, Jun 16, 2011 at 12:52 PM, aaleshin aales...@burnham.org wrote: I also like our Nanodrop, but I do not recommend using it for Bradford measurements. The 25% accuracy mentioned by Flip is pretty good for biological samples. Using 50 ul cuvette in a traditional spectrophotometer will not give this accuracy because cleanness of the cuvette will be a big issue... Alex On Jun 16, 2011, at 12:43 PM, Oganesyan, Vaheh wrote: I completely disagree with Filip’s assessment. I’ve been using nanodrop nearly 5 years and never had inconsistency issues. If you work at reasonable speed (if you put a drop there then lower the lever and click measure before you do anything else) there will be no issues. At very high concentrations the accuracy and therefore consistency may become lower. Concentrations between 5 and 10 mg/ml should be fine. The instrument is pricey though. * Vaheh* -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Filip Van Petegem *Sent:* Thursday, June 16, 2011 3:34 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford. Dear Arnon, the Bradford method is not recommended for accurate measurements. The readings are strongly dependent on the amino acid composition. A much better method is using the absorption at 280nm under denaturing conditions (6M Guanidine), and using calculated extinction coefficients based on the composition of mostly Tyrosine and Tryptophan residues (+ disulfide bonds). This method is also old (Edelhoch, 1967), but very reliable. One thing about the nanodrop: smaller volume = more evaporation. On the demo we've had, I was so unimpressed with the precision (25% variability between two consecutive measurement) that we didn't consider this instrument at all. So unless you just want a 'rough' estimate, I wouldn't recommend it at all. But most respectable spectrophotometers will take cuvettes with 50ul volumes - a big step up from 1ml volumes... Filip Van Petegem On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie la...@uic.edu wrote: Dear fellow crystallographers - a question about spectrophotometers for protein concentration determination. We are so last millennium - using Bradford reagent/ 1 ml cuvette for protein conc. determination. We have been considering buying a Nanodrop machine (small volume, no dilution needed, fast, easy). However, while testing our samples using a colleague's machine, we have gotten readings up to 100% different to our Bradford assay (all fully purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. So while it is fun/easy to use the Nanodrop, I am not sure how reliable are the measurements (your thoughts?). So QUESTION 1: What are people's experience regarding the correlation between Nanodrop and Bradford? While researching the Nanodrop machine, I heard about the Implen NanoPhotmeter Pearl. So Question 2: Is the Pearl better/worse/same as the Nanodrop for our purpose? Thank you for helping us to advance to the next millennium, even if it is nearly a dozen years late. Arnon -- *** Arnon Lavie, Professor Dept. of Biochemistry and Molecular Genetics University of Illinois at Chicago 900 S. Ashland Ave. Molecular Biology Research Building, Room 1108 (M/C 669) Chicago, IL 60607 U.S.A. Tel:(312) 355-5029 Fax:(312) 355-4535 E-mail: la...@uic.edu http://www.uic.edu/labs/lavie/ *** -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication
Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.
Hello Justin and others, The volume comment I make is based on mixing prior to the experiment, e.g. with Bradford reagent, Guanidine for the Edelhoch method, etc. Any direct measurement of A280 of protein samples requires you to know the extinction coefficient, which depends on the amount of Tyrosines and Tryptophans in the protein, but *also* the folding, as shown almost 5 decades ago. One way of minimizing this error is denaturing your protein with e.g. 6M Guanidine, at which point the extinction coefficient becomes largely independent of the protein composition, resulting in a more accurate concentration determination.( http://www.chem.uky.edu/courses/che554/Photometry/Edelhoch1967.pdf ) Bottom line: if you just put your protein on the nanodrop, and rely on the A280 absorption, there will be a systematic error. If you want to use e.g. Edelhoch's method, or any other method that requires mixing, the error will be larger for smaller volumes. cheers Filip Van Petegem On Thu, Jun 16, 2011 at 2:08 PM, Justin Hall hallj...@onid.orst.edu wrote: Hi Alex, I read Filip's comment about volume not as a path length argument, but about concentration uncertainty in mixing small volumes to dilute a sample down before measuring it (?). I have never had to make a dilution for my nanodrop (my proteins are usually not that concentrated), but I could see his point if I did have to. As for the variance between samples, I don't know about 25%, but I have observed multiple readings to have variance. I always take 3 readings on my nanodrop and then average them to deal with the variance I see. I don't mind doing this because the instrument is so fast, and I don't mind the cost at 6 ul of sample total. The most variance I have seen is usually in spin columns, where I will be doing a buffer exchange from a storage buffer (sometimes at ca. 20% glycerol) into an assay or xstal buffer, and I have wondered to myself if the variance I see could be due to incomplete mixing of a protein sample betwen a viscous buffer at the bottom with the rest of the buffer. I don't know how often other people find themselves in a situation where they may be sampling their 2 ul from a micro-environment that is not homogenous with the rest of the sample, but with small volumes I think that be a problem. Food for thought. Filip, I would buy a nanodrop. It is much better than a Bradford/cuvette and your students will love you for it. Cheers~ ~Justin Quoting aaleshin aales...@burnham.org: Filip, 25% accuracy is observed only for very diluted (OD280 0.1) or concentrated samples. But those sample a rarely used for ITC or CD. The concentrated samples require dilution but a regular spec does it too. Since the light passway is very short in Nanodrop it is accurate with more concentrated samples, which we crystallographers use, so Nanodrop is ideal instrument for our trade. If the drop is within recommended volume like 1-2 ul for our model, its size has a very small influence on the measurement. Cuvettes will give a better accuracy provided you clean them properly. I hated those times when I had to measure a concentration because of a need to wash a cuvette. In a biological lab they are always dirty. We switched to plastic disposable cuvettes for that reason... Alex On Jun 16, 2011, at 1:06 PM, Filip Van Petegem wrote: 25% is not acceptable for ITC or CD experiments though... I was just sharing our bad experience with a demo nanodrop we had. Even if evaporation is not an issue, one has to take pipetting errors into account when dealing with small volumes. The relative error on 1-2ul is a lot bigger than on 50ul. Unless you want to pre-mix 50ul and use a small quantity of that, which defeats the purpose of miniaturization... It all depends on your applications and sample availability, but if you want a very accurate measurement, miniaturized volumes just won't get you the same accuracy. Cuvettes will give a better accuracy provided you clean them properly. Just some water or EtOH is *not* enough... Filip Van Petegem On Thu, Jun 16, 2011 at 12:52 PM, aaleshin aales...@burnham.org wrote: I also like our Nanodrop, but I do not recommend using it for Bradford measurements. The 25% accuracy mentioned by Flip is pretty good for biological samples. Using 50 ul cuvette in a traditional spectrophotometer will not give this accuracy because cleanness of the cuvette will be a big issue... Alex On Jun 16, 2011, at 12:43 PM, Oganesyan, Vaheh wrote: I completely disagree with Filip’s assessment. I’ve been using nanodrop nearly 5 years and never had inconsistency issues. If you work at reasonable speed (if you put a drop there then lower the lever and click measure before you do anything else) there will be no issues. At very high concentrations the accuracy and therefore consistency may become lower. Concentrations between 5 and 10 mg/ml should be fine
Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.
A convenient fast way is the earlier mentioned Edelhoch method, as described in this paper which is referenced on the popular Protparam tool: http://onlinelibrary.wiley.com/doi/10.1002/pro.5560041120/pdf Filip On Thu, Jun 16, 2011 at 4:45 PM, aaleshin aales...@burnham.org wrote: Mischa, You intrigued me. What is the experimental technique for the Extinction Coefficient measurement (which requires knowledge of protein concentration)? Let me guess, Bradford? Protein evaporation and weighing? Alex On Jun 16, 2011, at 4:22 PM, Machius, Mischa Christian wrote: With respect to the Edelhoch method and the ProtParam server, I would strongly recommend determining extinction coefficients experimentally and not rely on the ProtParam values. The reason is that the underlying extinction coefficients in the formula used by ProtParam and referenced there are statistical averages. They may or may not be valid for a given protein. I have seen differences of more than 20% between the theoretical and experimental extinction coefficients, particularly for proteins with few Trp and Tyr residues. When relying on relative concentrations, this inaccuracy is not detrimental, but when absolute concentrations are needed (CD, AUC, ITC, any binding experiment, etc.), such a difference would be considered huge. Determining an extinction coefficient experimentally takes but a few minutes. Cheers! MM On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote: Totally support the statements below. We have had several proteins with A280 absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in the Nanodrop or whatnot to measure the concentration. Before purchasing the Nanodrop we used a Hellma TrayCell and a normal UV/Vis instrument. Similar to the Nanodrop, the sample volume in TrayCell is 2-3 ul. Traycell works well at a fraction of the Nanodrop cost, but Nanodrop is a lot more convenient to use for high concentration quick measurements (especially if you need to measure several things in succession), so you get what you pay for. Petr P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). That plus the Nanodrop are two essential and synergetic tools of a protein chemist/crystallographer. On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote: Bradford is an assay, Nanodrop is a spectrophotometer. Both the A280 and Bradford methods are strongly dependent on amino acid composition, so unless you correct A280 for that as mentioned by Filip, either one is semiquantitative. Occasionally you come across a protein with no tryptophan which will have a much lower extinction coefficient. Try making a 1 g/l solution of gelatin (collagen?) and see what its A280 is! I noticed recently the protparam tool at http://ca.expasy.org/cgi-bin/protparam estimates the extinction coefficient given a sequence. David Briggs wrote: ~~~ I wouldn't touch Bradford with a barge-pole. I've found it to be wildly inaccurate for certain proteins I've handled, where as the OD280 measurements have been fine. One wonders what does fine mean, like same as with Biuret or Kjeldahl nitrogen, or solution made up by weight? --- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485 fax: +1-919-966-5640 email: mach...@unc.edu mach...@med.unc.edu -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] EM map sigma level.
Dear Hailiang, most maps (if not all) have the suggested contour level associated with them in the visualization tab of their EMDB entry. It is my understanding that this is the contour level that will account for the Van Der Waals volume of the protein. The Download tab allows you to download a CCP4-style map, which you can read in with your favorite molecular graphics package. Filip Van Petegem On Thu, Jun 2, 2011 at 2:24 PM, Hailiang Zhang zhan...@umbc.edu wrote: Hi, I am trying to compare a published EM map with X-ray map in hand, and have several questions: 1. EM map seldom indicates the sigma level, and it was said because of the box size uncertainty during EM model construction. Now, I wonder is there any way we can sort of its equivalent sigma level to X-ray map? 2. The EM structure deposited in pdb don't have any experimental data, and not sure how to obtain them and generate the map file. Sorry if this is wrong place to post EM questions. Hailiang -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] 250 kDa standard
But this wouldn't correspond to a globular form; 250kDa DNA will appear larger than a 250kDa globular protein. On Sat, Feb 19, 2011 at 5:25 PM, Artem Evdokimov artem.evdoki...@gmail.comwrote: A sufficiently long piece of DNA works OK. On Sat, Feb 19, 2011 at 6:47 PM, Alexandra Deaconescu deac...@brandeis.edu wrote: Dear ccp4bb enthusiasts: A question unrelated to ccp4: can anyone recommend a good 250 kDa standard for gel filtration that is commercially available? It could be a single polypeptide or an oligomer too... Thanks a lot! Bests, Alex -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
[ccp4bb] Low number of waters for a given resolution
Hello all, Some crystal structures seem to contain a very low number of water molecules given their resolution (e.g 1 water for every 10 amino acid residues at 2.2Angstrom, whereas probably around 0.6 waters per residue is expected on average). I was wondering if anybody has any insights (or better: a good reference) into the precise reasons. Of course general data quality comes into mind, or using data-to-parameter ratio rather than resolution. But how about intrinsic properties the protein? So my exact questions are: - How frequent is a very low number of visible waters observed? - What are the usual reasons? Cheers Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] protein concentration in crystal
Hello Teresa, c = x/(N.V) where x=number of molecules per unit cell, N=Avogadro's number, V=volume of the unit cell in liter, c=concentration in molar Cheers Filip On Mon, Dec 13, 2010 at 12:07 PM, Teresa De la Mora dela0...@umn.eduwrote: Hi all I'm looking for a way to calculate the protein concentration in a single crystal. So what I'm thinking is to use the Matthew's number to calculate how many molecules inside the crystal, then multiply that number by Avogadro's number to get moles then divide by volume of crystal. Is this approach correct? Thank you for your advice/suggestion Happy holidays! :) Teresa Teresa De la Mora-Rey Ph.D. Dept. Medicinal Chemistry University of Minnesota 8-101 Weaver-Densford Hall 308 Harvard St. SE, Minneapolis, MN 55455 Lab phone (612) 626-5226 If you never did you should. These things are fun and fun is good Dr. Seuss -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] how to optimize small rod-shaped crystals
One problem with a dye like methylene blue is that it tends to crystallize in certain conditions commonly found in crystallization screens (e.g. some that are high in PEG) making them less useful in such conditions. Has anybody systematically tested alternative dyes and found one that is more soluble? Cheers Filip Van Petegem On Tue, Nov 16, 2010 at 6:15 PM, Jim Pflugrath jim.pflugr...@rigaku.comwrote: With Izit or other dyes, you might wish to do a positive control with bona fide protein crystals and a negative control with bona fide salt crystals. -- *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Matthew Bratkowski *Sent:* Tuesday, November 16, 2010 7:58 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] how to optimize small rod-shaped crystals I like using Izit dye from Hampton ( http://hamptonresearch.com/product_detail.aspx?cid=4sid=41pid=33) to check if crystals are protein or salt. If the crystals are protein, the dye should absorb rather readily into the crystals and turn them blue, while the rest of the drop will eventually turn clear. Quite likely, excess dye will also crystallize out as well. Salt crystals will not soak in the dye, and the rest of the drop may remain blue for several days. Using Izit is easy and saves a lot of time. In my experience, I have gotten a lot of false positives from phosphate crystallization conditions, so you want to be sure that the crystals are not salt before you waste any time on optimizing them. Matt -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] precipitation after storage
Hello Eric, Does your protein also precipitate at lower protein concentrations? In isolated cases, we've had protein stocks precipitate overnight at 4 degrees, and the only way around it was to store them diluted, and concentrate right before any experiments/crystallization trials. In two cases, the 'precipitation' in the protein stock appeared to be showers of microcrystals... Filip On Fri, Nov 5, 2010 at 1:40 PM, Eric Karg harvard...@yahoo.com wrote: Dear all, I'm working on a protein which starts to precipitate after 3-4 days of storage at 4 degrees or room temperature. The storage buffer contains 300 mM NaCl because at lower salt concentrations it also tends to precipitate. Different buffers and adding glycerol did not help although this was not done in a systematic way. Has anyone had similar experiences? Any suggestions to overcome this problem? Thanks in advance! Eric -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] protein turns brown
I also have a similar observation for proteins purified by Ni-NTA column. After concentrating the sample eluted from the Ni-NTA column, I see a brownish-yellowish tinge closer to the bottom of the filter with colorless buffer on top. This is observed even for non-metal binding and non-FeS cluster proteins and also for proteins after removal of the His-Tag. This is presumably arising due to difference in viscosity where the protein gets concentrated closer to the bottom of the filter. Or more likely: Nickel bleeding from the column was complexing bME or DTT in your sample: Ni-bME forms a tight brown complex. Filip Van Petegem
Re: [ccp4bb] conversion of cyroEM reconstruction from MRC to CCP4 format
Hello James, You can try the map2map from the SITUS package ( http://situs.biomachina.org/fguide.html) or em2em ( http://www.imagescience.de/em2em/) Cheers Filip Van Petegem On Tue, Jul 13, 2010 at 5:32 AM, James Whittle whit...@crystal.harvard.eduwrote: Hi all- I'm trying to convert a cryoEM map from FREALIGN for use with various CCP4 programs, or with MAPMAN. Even though the MRC format is derived from the CCP4 map file format, every program I've tried fails to open it, (except for Chimera which displays the map but cannot save it to another format for lack of memory). For example, in MAPMASK I get the error: CCP4 library signal ccp4_map:No associated header (Error) raised in ccp4_cmap_open CCP4 library signal ccp4_map:Cannot open file (Error) raised in MRDHDS or with MAPMAN: **CCPOPN ERROR** FORMATTED OLD file open failure on unit 1 Logical name: DLP_100.map, File name: DLP_100.map Cannot send after transport endpoint shutdown ERROR --- While reading map header. Sorry ! ERROR --- While opening map file Is there a way to re-write the header to be compatible with CCP4? --James -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
[ccp4bb] stories on cryoEM frustration
Dear structural biologists, I'm sure some of you have been there before: you'd like to perform docking experiments with your favorite crystal structure in your favorite cryoEM map. Only problem is, the authors of the cryoEM study simply refuse to send you their maps, and they're not available in the EMDB database. I'm assembling a list of stories of frustration to be used at the upcoming USNCCr meeting. The idea is to get an overview of which research fields have these types of associated issues. I'll guarantee anonymity for anybody who sends me their story. I'd appreciate if you could send me the reference to the primary paper that described the cryoEM structure, as well as the specific issue and outcome (e.g. were the journal editors contacted and could they resolve the issue). Thanks Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] voltage gated Na channel
Dear Rex, It might still be while before a structure of a full-length voltage-gated sodium channel (or voltage-gated calcium channel) comes along. Keep an eye on the NaChBac channel (originating from Bacillus species), as this prokaryotic sodium-selective channel will likely be the first to represent the sodium channel family. (Science.http://www.ncbi.nlm.nih.gov/pubmed/11743207 2001 ;294,2372-2375) There are structures of small cytoplasmic loop segments (PDB ID 2KAV, 1BYY, 1QG9), but these won't tell you anything about sodium selectivity. cheers Filip Van Petegem On Wed, May 26, 2010 at 4:50 AM, Rex Palmer rex.pal...@btinternet.comwrote: Does anyone know of the existence of a structure for a voltage gated Na (not K) channel? Rex Palmer Birkbeck College, London -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] electron microscopy: where open access fails
Dear Cathy/EMDATABANK team: It is hard to comprehend the option for keeping maps on hold for up to 2 years. It seems any depositor would do this for pure selfish reasons: keep the data to themselves, don't allow anybody to verify the data for a long time, and have the exclusive right to do experiments with the maps. For example, this would allow the depositor to be the only one perform docking experiments with any partial crystal structure for 2 years, and also these experiments wouldn't be falsifiable for a period of 2 years (!). Comparing this with crystallography: one would keep a crystal structure of a good drug target 'on hold' for 2 years, thus not allowing anybody to use it to start rationally designing new drugs (the success rate aside for this matter). In scientific terms, two years is 'huge'. It is in this time frame that a new theory can be postulated by one, and then shot down by ten other papers. It is 40% of the time frame of an NIH ROI1 grant, and 66% of a typical Canadian CIHR grant. When it comes to cryoEM and crystal structures of important therapeutic targets, delaying the field for 2 years will ultimate cost lives. In the end, journal editors should create firmer and waterproof policies like those implemented for crystal structures, such as not allowing publication until the data are in the 'hold for publication' status. Some journals already have the clear policy of requiring deposition of the cryoEM maps, but the 2 year hold is currently a big loophole. Many more journals, however, don't require the deposition of maps at all. Of course it would help if the EMDB didn't allow for these loopholes... Sincerely, Filip Van Petegem On Thu, May 20, 2010 at 7:16 AM, Cathy Lawson cathy.law...@rutgers.eduwrote: sent on behalf of the EMDATABANK.org team: The EM Databank (EMDB, http://www.emdatabank.org/) is a resource for the archival deposition and retrieval of EM maps and associated metadata. It was established in 2002 by the European Bioinformatics Institute (EMBL-EBI, UK), and is now run jointly by EBI, the Research Collaboratory for Structural Bioinformatics (RCSB, USA), and the National Center for Macromolecular Imaging (NCMI) at Baylor College of Medicine. Following the model of the wwPDB, development of EMDB policies and procedures is community-driven. The resource is advised by a panel of leading experts. This fall, an Electron Microscopy Validation Task Force (EM VTF) will be convened to make recommendations as to how best to assess the quality of both maps and models that have been obtained from cryo-EM data. Its recommendations will form the basis for a validation suite that will be used for maps and models deposited in the appropriate databases (EMDB and PDB). As seen by the history of the PDB, journal requirements can greatly influence data deposition. For articles reporting the results of electron microscopy studies, the rate of EM map deposition is higher for journals that have well-defined and consistently-enforced policies than for journals without deposition requirements. We have recently contacted journals that publish EM studies to encourage them to include a deposition policy for EM structural data in the instructions to Authors, and we are continuing to follow up with them. Currently, depositors may choose to release deposited data immediately, upon publication (selected by the majority), after 1 year, or after 2 years. The 1 and 2 year holds are intended to encourage EM scientists to deposit maps by providing a time period in which they can perform additional studies/analyses before the map is made public. Based upon community feedback, the option to hold a map for 4 years was retired in 2008. Questions about the EMDB may be sent to h...@emdatabank.org. -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] electron microscopy: where open access fails
Dear crystallographers, For those of you who have shared personal frustration with cryoEM map availability, or for those of you who would simply like to see science proceed as it should, here's your opportunity to sign an on-line petitition. Please feel free to send the link below to any of your colleagues. More signatures = more pressure. http://www.petitiononline.com/cryoEM/petition.html Sincerely, Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
[ccp4bb] electron microscopy: where open access fails
Dear colleagues, whereas data sharing for most crystallographers appears to be a no-brainer, making coordinates and (most of the time, hopefully) structure factors available, it seems the electron microscopists are drastically lagging behind when it comes to making data available. Many cryoEM structures are still being published without the corresponding maps being deposited in the EM database. In one particular case, I was interested in looking at a cryoEM map from a paper published in a well-renowned open access journal starting. The paper contains the EMDB accession codes for the maps, but these maps appear to be 'on hold' since over a year. Enquiry with the authors delivered a firm 'no' in releasing the maps: they claim it is OK to keep the maps on hold for 2 years, simply because the EMDB gives the option to do so. A further enquiry with the journal editors led to no avail: despite the clear journal policy on sharing both manuscripts and data, they were also unable to force the authors to release their maps, now ~13 months after publication of the paper. The fact that this was in an open access journal makes this all the more shocking. It is striking to see how much still has to be done to lift the cryoEM world up to the same standards as the crystallographic community (when it comes to sharing data, at least). Structures can simply be published without anybody being able to check the validity, let alone use it for obvious experiments such as docking crystal structures, integrative protein structure modeling, etc. With many structural targets going towards bigger and more challenging, combining cryoEM data with crystal structures is going to become more and more important. What can we, crystallographers, do to stimulate data-sharing in the cryoEM world? (My apologies to the cryoEM people on this bulletin board: if you have been making your maps available, you'll agree that clearly not everybody does...) Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] very high concentration of protein
Dear Peter, it's a common phenomenon to create protein concentration gradients inside a protein concentrator. Simply take it all out of the concentrator, put it in a separate tube, and mix thoroughly/vortex. The 'slime' may very well redissolve and you'll a homogeneous distribution. What you describe happens a lot to us with proteins that don't precipitate and that crystallize afterwards. Cheers Filip Van Petegem On Thu, Jul 2, 2009 at 8:53 AM, peter hudson peter.hudson.pe...@gmail.comwrote: Hello all I am working with a small protein-protein complex. This complex express quite well . I purify in a buffer of pH=9.0 with 150mM NaCl and 1% of glycerol and able to concentrate upto 20 mg per ml. I have a two clones of this protein complex. One is N-terminal His tagged and another C-terminal His-tagged. While concentration of the N-terminal His tagged protein in cnetricon it forms yellow color slimy deposition on the surface of membrane. while C-terminal His tagged protein does form very highly viscous layere at the surface of membrane but it is completly colourless.I aliquate the concentrated protein by pippetting into different aliquate rather than collection of whole protein by centrifugation. if i check the concentration of the last aliqoute(which isbottommost viscous protein part) both prtoein complex, it shows very high concentration compared to the first fraction. I have not done DLS. Is my both C-terminal His tagged tagged as well as N-terminal His tagged protein are forming soluble aggregates. I would appreciate the help. Thanks in advance Peter -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] precipitation of deglycosylated protein
Dear Simon, this may be an isolated case, but you might want to try to drastically change the pH of the reaction conditions. In our hands, a protein with 3 hyperglycosylated sites, expressed in Pichia pastoris, could be deglycosylated readily with endoH. However, at the recommended reaction pH (5-5.5), the protein rapidly precipitated in an irreversible manner. After testing the reaction in various different buffers, nearly all of the protein could be deglycosylated at pH 7.5, with no visible precipitation. Curiously, the buffer could easily be exchanged to pH 5.5 and below after the reaction was finished (the protein even crystallized at pH 4). This of course only has a small chance of working in your case, but it's quick to test various different mini-deglycosylation experiments using an array of conditions. Cheers Filip Van Petegem On Mon, Mar 16, 2009 at 9:29 AM, Yue Li simon.yu...@yahoo.com wrote: Hi everyone, Recently, I obtained a soluble glyco-protein. Unfortunately, after I added PNGase or Endo Hf to remove the glycans, the deglycosylated protein is precipitated. Is there any method to avoid this kind of precipitation? Thanks, Simon -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] cryoloops for X-ray data collection from protein crystals at room temperature
Dear Cedric, We use a much easier test for mounting crystals at room temperature: just coat the crystal with paratone oil and mount your crystal in a standard cryoloop. The oil will slow down evaporation enough - no special tools required. You don't even need to remove all the liquid as you would do for flash-cooling the crystal in paratone. And a major advantage: you can use the same crystal to collect under cryo-conditions and directly compare the impact of cooling the crystal... Cheers Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ On Fri, Jan 16, 2009 at 6:27 AM, cedric bauvois cbauv...@gmail.com wrote: Dear CCP4ers, in their paper entitled Using cryoloops for X-ray data collection from protein crystals at room temperature: A simple applicable method ( *Journal of Crystal Growth* http://www.sciencedirect.com/science/journal/00220248 Volume 281, Issues 2-4http://www.sciencedirect.com/science?_ob=PublicationURL_tockey=%23TOC%235302%232005%23997189997%23601824%23FLA%23_cdi=5302_pubType=Jview=c_auth=y_acct=C26678_version=1_urlVersion=0_userid=532047md5=9a4e7b2fc158c6d2396925c79d995e3d, 1 August 2005, Pages 592-595.), the authors present a way to mount crystals using a cryoloop accompanied by a glass capillary cap (see abstract below). Do you know if any commercial version of such system are now available ? Abstract: Although cryoloops are now routinely used for X-ray data collection from protein crystals in cryocooling condition, it is still necessary to collect X-ray diffraction data from protein crystals at room temperature under such circumstances as to find resolution limit and/or to avoid damage of protein crystals at cryogenic temperature (e.g. 100 K). Here, we show that a cryoloop, which is accompanied by a glass capillary cap to maintain humid environment of crystal in the cryoloop, can be used not only to examine protein or non-protein crystals but also to collect X-ray diffraction data for structural analysis from protein crystals at room temperature. The size of cryoloop should be carefully chosen so that the crystal does not move in the cryoloop. This crystal mounting method can be time-saving compared to the traditional method to mount a crystal in a glass capillary tube. Many thanks -- Dr. Cedric Bauvois Cristallographie des protéines Institut de Recherches Microbiologiques JM Wiame -IRMW Av E. Gryzon 1, 1070 Brussels (Belgium) tél: +32 (0)2 5273634 fax: +32 (0)2 5267273
Re: [ccp4bb] suggestions for UV spectrometer
I want to add I absotely hate the nanodrop. We've had a demo for it, and found the readouts to be very unreliable. Fluctuations of 20% and more. Just leaving the same drop in and measuring the sample multiple times gives different values (going in both directions, so not only due to evaportations). Sure, it's easy and fast, and maybe good to have a rough idea about your protein concentration, but I would never want to use it for exact measurements such as needed for e.g. a CD or an ITC instrument. I've heard other labs in our department have similar issues. We've also had a demo for the Nanovue from GE Healthcare: same issues - very large fluctuations from one sample to another. I suppose this is simply an inherent problem with small volumes... Cheers Filip Van Petegm On Thu, Dec 4, 2008 at 12:48 PM, Patrick Loll [EMAIL PROTECTED] wrote: At the risk of dragging this discussion even further afield from crystallography: How can you get realistic numbers for concentrated solutions using the Nanodrop? I understand that the instrument reduces absorbance by using a very short path length. However, I thought that in order for the Beer-Lambert formalism to be applicable, the solution needs to be sufficiently dilute so that the chance of molecules shadowing one another is negligible. Isn't this condition violated for concentrated solutions (even with short path lengths)? Pat On 4 Dec 2008, at 1:27 PM, Michael Giffin wrote: We also like the Nanodrop... --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 [EMAIL PROTECTED] -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Protein Color
Hello Matt, did you use a nickel (or similar) affinity column in the purification + is there any beta-mercaptoethanol or DTT in your sample? Nickel can easily 'bleed' off the column and will turn brown when complexed with bME or DTT - this complex can get quite large and even insoluble if enough is present, and would therefore concentrate in your sample. Cheers Filip On Sat, Sep 6, 2008 at 4:49 AM, Joe Cockburn [EMAIL PROTECTED] wrote: Hi Matt, I sometimes see a similar thing with my proteins, which definitely don't possess metal co-factors or prosthetic groups. I found that gel filtration got rid of it - the browny-yellow stuff came out in the void fraction so I figured it was aggregated protein. I think it was aggregation via the his-tags around traces of copper in my sample, which could explain the brown-ish colour. What happens if you concentrate the protein in the presence of EDTA? Joe Hello. I am working with a protein that turns a yellowish-brown color when it is concentrated to around 2 mg/ml or higher in a small volume (a few hundred uL). I was wondering if the protein bound a metal or other prosthetic group that would give it this color? The protein's color somewhat resembles iron binding proteins, but there is no peak in the 400 nm range that would suggest heme, and an iron sulfur cluster is not that likely since there are only five cysteines in the protein. Proteins with structures homologous to the one I am studying bind magnesium, but are not know to bind other metals. Any information about what this color might suggest about the protein or how I could analyze possible bound metals or prosthetic groups using only a small amount of protein would be helpful. Matt -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Puzzling protein-protein interaction
Dear Brett, since your deltaH contribution gets weaker, and the deltaS contribution changes from negative to neutral, you could have a case of enthalpy-entropy compensation (see e.g.Dunitz JD (1995) Win some, lose some: enthalpy-entropy compensation in weak intermolecular interactions. *Chem Biol* 2, 709–712). As for the discrepancy between the pull-down and the ITC, remember that a pull-down is not an equilibrium measurement. If you keep washing long enough (hours, days, months), you will eventually lose all binding. The length of time required to wash it off is partly dependent on the kinetics of binding and unbinding. SPR could answer if that's the case. Other things to consider: temperature difference between ITC and pull-down? Cheers Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/ On Tue, Sep 2, 2008 at 1:37 PM, Brett Collins [EMAIL PROTECTED]wrote: Dear CCP4 Community, My apologies for the non-crystallography biochemical question but it occurred to me that there are many people on this list who are also very good biochemists. We have just performed an ITC experiment with two proteins and measured a Kd of 150 nM, deltaH of -15 kCal/mol, deltaS of -15 Cal/mol/K and deltaCp of -2000 J/Mol/K. We also measured the binding of a mutant of one of these proteins predicted from crystal structure to inhibit binding of a small fragment of peptide (this is predicted to reduce binding slightly but not to affect total binding as there is still a large interaction interface that is left intact). This mutant has a Kd of 150 nM as well, but deltaH is -10 kCal/mol, delta S is essentially zero, and deltaCp reduces in magnitude to about -1500 J/Mol/K as we would predict from the change of buried surface area. The ITC data looks good and we have repeated the experiments a number of times so they are statistically significant. The experiments were performed within reasonable concentration limits (~10uM protein in the cell so the C-value is about 50-100) Now the puzzle is that the mutant binds less strongly in pulldowns (about 50% reduction after several washes) but we see an almost identical Kd by ITC despite major changes in enthalpy/entropy contributions to binding. The mutant and wildtype appear to have identical fold by CD but of course there may be small differences. Everything makes sense except the lack of Kd change by ITC. Does anyone have any experience of similar results, or more importantly have a possible explanation for them? Any thoughts greatly appreciated. Brett Collins
Re: [ccp4bb] peptide for co-crystallization
Hello Daniel, on the note of peptides being more pure than the minimum, I've had the reverse experience. A 20aa peptide I ordered was 'claimed' to be 90% pure; the order included MALDI and HPLC profiles to 'prove it', but after running an HPLC myself, found 3 peaks equal in size, and only one of them had the right mass. I won't tell the name of the company, but it's worthwhile to check the purity for yourself. Cheers Filip On Wed, Aug 27, 2008 at 10:04 AM, Daniel Jin [EMAIL PROTECTED] wrote: Hi, We would like to order two peptides for co-crystallizaiton. I notice that there is a big price jump for peptide over 95% pure and over 98% pure. Do you think 95% purity is good enough? Another choice is to order crude peptides and purify by ourselves. Crude peptide is much cheaper. But we will then have to put in time to do multiple HPLC runs, reagent, cost for Mass Spec verification... Does anyone have suggestion about which one will be more efficient? Thanks. Best, Chen -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Weakest protein-protein complex crystallised
Hi, we've had a similar situation: a protein-peptide complex with a Kd in the nM range crystallized in the same condition as the protein alone, and yielded a structure of a complex (voltage-gated calcium channel beta subunit). The exact crystal contacts turned out to be a bit different, as the peptide would clash with a neighbouring molecule in the lattice. However, a mutant protein that increased the Kd to ca 160nM (as confirmed by ITC), using the same peptide crystallized in the same conditions, but this time not as a complex. This effect was reproducible: the WT consistently crystallized as complex, whereas relatively mild mutants (Kd in 100nM range and worse) only yielded crystals of the apo-protein. Conclusion would be that crystal contacts can break relatively tight protein-protein interactions in the ~100nM range, and that crystal contacts are not always that weak. However, the crystallization conditions themselves (PEGs, non-neutral pH) are likely to affect the binding as well. Cheers Filip Van Petegem On Mon, Jun 30, 2008 at 10:42 AM, Philippe DUMAS [EMAIL PROTECTED] wrote: Hello We have had an interesting example where the crystal packing seems to have won against the biological interaction. This is about a sliding clamp made of a very symmetric homodimer having the shape of a ring (encircling DNA during its replication). This beta-ring had been crystallized alone by the Kuriyan group in P1 (thus there was NCS). In our case, we crystallized it with an additional peptide mimicking the C-term of a polymerase binding to the beta-ring [Burnouf et al, JMB 335(2004) 1187]. We expected a symmetric binding of two peptides/ring (one peptide for each protein in the dimer). However, we observed only one peptide/ring. It turns out that we had obtained exactly the same packing in P1 and that one of the two possible binding sites was engaged in crystal contacts. We estimated the Kd of peptide-ring interaction as being in the µmolar range and that there was only a few percent of beta-rings in crystallization drops being singly occupied. Yet the crystallization process selected this minor species to build crystals with (supposedly) a good crystal packing, rather than finding another crystal packing accomodating the doubly-occupied species present in large excess. Our conclusion was that a very modest gain of ca. 2 kcal/mol in the free energy of interaction of singly-occupied beta-rings was sufficient to account for their selection to build crystals against a great majority of doubly-occupied contaminants. This is exactly the order of magnitude mentioned by Ed Pozharski: a single additional H-bond is enough to account for 2 kcal/mol ! And apparently this may be enough to win against biological interactions. Let us not forget that there are many processes comparable to crystallization in living cell... I hope this story makes sense in the frame of this discussion. Philippe Dumas IBMC-CNRS, UPR9002 15, rue René Descartes 67084 Strasbourg cedex tel: +33 (0)3 88 41 70 02 [EMAIL PROTECTED] -Message d'origine- De : CCP4 bulletin board [mailto:[EMAIL PROTECTED] la part de Ed Pozharski Envoyé : Monday, June 30, 2008 4:50 PM À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] Weakest protein-protein complex crystallised The word weak is, of course, relative. Free energy of crystallization is roughly 1-2 kcal/mole of crystal contacts (I think I carried this number from Sir Blundell's book, but quick look at papers by Peter Vekilov's group seems to confirm it - am I wrong on this?). I think that crystal contacts are still much weaker than any interaction of biological importance (perhaps I am wrong on this one too and there are important biological protein-protein interaction with 10mM affinity, but I doubt that they are many). On Mon, 2008-06-30 at 10:09 -0400, Patrick Loll wrote: I hope this isn't too much of a foray into philosophy and semantics, but can't you argue that the crystals themselves are weak complexes? And since the energies of crystal contacts are typically very weak, I would further argue that you should be able to crystallize ANY complex with an association constant corresponding to energies as low as those associated with crystal contacts. Of course, it's not guaranteed, any more than getting a crystal is guaranteed--you need some luck. Of course, it's Monday AM, and I haven't approached my asymptote for caffeination. Am I talking through my hat? Pat On 29 Jun 2008, at 3:36 PM, Derek Logan wrote: Hi, Can anyone advise me what is currently the weakest protein-protein complex yet crystallised? Google searching turned up a paper from the Tromsø crystallography group (Helland et al. 1999, JMB 287, 923– 942) in which a complex between beta-trypsin and a P1 mutant of BPTI with a Kd of 68 uM was described as belonging to the weakest complexes solved to date, but this article
Re: [ccp4bb] Weakest protein-protein complex crystallised
Dear Jens, There is an overwhelming amount of evidence that our mutants still binds to the same site in solution and in vivo - there is a correlation between decrease in binding affinity (calorimetric measurements) and function (electrophysiological measurements) for more than 20 mutants based on the structure. The native binding site was found in 3 different crystal structures from independent groups (all different crystallization conditions and different variants). A crystal artefact of the observed WT binding is therefore excluded. The crystal artefact is that we don't observe any binding in the crystal structures of a set of mutants (neither to the native site, nor to any other), whereas both calorimetric and electrophysiological data suggest there should be binding in the 100-200nM range. The binding is abolished because of crystal contacts (+ crystallization conditions) for 100nM and weaker binders, but not for 10nM and stronger binders. What you were saying is the other way around (tight binders not binding). Cheers Filip Van Petegem On Mon, Jun 30, 2008 at 2:22 PM, Jens T. Kaiser [EMAIL PROTECTED] wrote: Dear Filip and others, To play Devils advocate, this could also (in the absence of strongly supportive biochemical data) be interpreted as a crystal artifact, with the weakly binding ligand not forming a physiologically relevant contact but merely occupying the - haphazardly - empty space in the crystal of the bigger protein. When tighter binders bind to the 'real' interaction side, they inhibit the crystal packing, thus yielding only crystals of the free partner. I'm not saying this is the case in your example, but this should be strongly considered for weak interactors (see for example the HLSV/U debate a few years ago, where, as far as I can remember, the regulator bound 'its' protease not in the 'regulating' conformation, but in a 'substrate' like fashion.) Cheers, Jens On Monday 30 June 2008 12:01:12 Filip Van Petegem wrote: Hi, we've had a similar situation: a protein-peptide complex with a Kd in the nM range crystallized in the same condition as the protein alone, and yielded a structure of a complex (voltage-gated calcium channel beta subunit). The exact crystal contacts turned out to be a bit different, as the peptide would clash with a neighbouring molecule in the lattice. However, a mutant protein that increased the Kd to ca 160nM (as confirmed by ITC), using the same peptide crystallized in the same conditions, but this time not as a complex. This effect was reproducible: the WT consistently crystallized as complex, whereas relatively mild mutants (Kd in 100nM range and worse) only yielded crystals of the apo-protein. Conclusion would be that crystal contacts can break relatively tight protein-protein interactions in the ~100nM range, and that crystal contacts are not always that weak. However, the crystallization conditions themselves (PEGs, non-neutral pH) are likely to affect the binding as well. Cheers Filip Van Petegem On Mon, Jun 30, 2008 at 10:42 AM, Philippe DUMAS [EMAIL PROTECTED] wrote: Hello We have had an interesting example where the crystal packing seems to have won against the biological interaction. This is about a sliding clamp made of a very symmetric homodimer having the shape of a ring (encircling DNA during its replication). This beta-ring had been crystallized alone by the Kuriyan group in P1 (thus there was NCS). In our case, we crystallized it with an additional peptide mimicking the C-term of a polymerase binding to the beta-ring [Burnouf et al, JMB 335(2004) 1187]. We expected a symmetric binding of two peptides/ring (one peptide for each protein in the dimer). However, we observed only one peptide/ring. It turns out that we had obtained exactly the same packing in P1 and that one of the two possible binding sites was engaged in crystal contacts. We estimated the Kd of peptide-ring interaction as being in the µmolar range and that there was only a few percent of beta-rings in crystallization drops being singly occupied. Yet the crystallization process selected this minor species to build crystals with (supposedly) a good crystal packing, rather than finding another crystal packing accomodating the doubly-occupied species present in large excess. Our conclusion was that a very modest gain of ca. 2 kcal/mol in the free energy of interaction of singly-occupied beta-rings was sufficient to account for their selection to build crystals against a great majority of doubly-occupied contaminants. This is exactly the order of magnitude mentioned by Ed Pozharski: a single additional H-bond is enough to account for 2 kcal/mol ! And apparently this may be enough to win against biological interactions. Let us not forget that there are many processes comparable to crystallization in living cell
Re: [ccp4bb] Weakest protein-protein complex crystallised
Hello John, No, they're not. Crystals were obtained at pH8.0, 200mM NaCl; 10% PEG4000. Calorimetric experiments were done at pH7.4, 150mM KCl. We found the interaction to be driven mainly by hydrophobic contacts (mutants of polar/charged residues have no significant effect on the affinity). I'd only expect a minor effect of pH in this case, but this would have to be tested. So yes, as I've mentioned before, both crystal contacts and crystallization conditions could together reduce the affinity and break the 100nM Kd interaction. The effect of pH on the affinity could be tested directly in an ITC experiment - the effect of 10% PEG4000 would be harder to assess due to insolubility (crystallizability) of the protein... Cheers Filip Van Petegem On Mon, Jun 30, 2008 at 4:47 PM, John A. Newitt [EMAIL PROTECTED] wrote: At 3:28 PM -0700 6/30/08, Filip Van Petegem wrote: The crystal artefact is that we don't observe any binding in the crystal structures of a set of mutants (neither to the native site, nor to any other), whereas both calorimetric and electrophysiological data suggest there should be binding in the 100-200nM range. The binding is abolished because of crystal contacts (+ crystallization conditions) for 100nM and weaker binders, but not for 10nM and stronger binders. Filip: Are you calorimetric binding measurements performed under similar conditions (especially pH) as your crystallization condition for the mutant proteins? We have determined in some cases that apo crystals are due to the fact that a ligand had reduced affinity at the non-neutral pH of crystallization, whereas initial positive binding studies were performed at pH ~7. - John -- http://xri.net/=john.newitt -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Dnase activity in E. coli expressed his tag proteins
Dear Narayanan, you could simply test that by a negative control: transform your cells with an empty vector, and follow the same (exact) purification protocol. No DNase activity there = probably no DNase contaminant in your original prep. Cheers Filip On 8/6/07, Narayanan Ramasubbu [EMAIL PROTECTED] wrote: Dear All: This is off topic but I would rather try here first. I am wondering whether Dnase could be a contaminant during the nickel affinity purification of a his.tag protein expressed using pET29b. The cells were disrupted using sonication only. Very high yield (30 mg/liter of cell culture). FPLC purification. SDS-PAGE shows a singe band even when overloaded. Silver staining is in progress. The protein I am interested in does not have any similarity to Dnase. However, when treated with supercoiled dna, the dna is degraded as efficiently as with Dnase I from Sigma. I am looking for ways to show that my preparation does not have any Dnase contamination. I would appreciate it very much if someone points me in the right direction. Thanks a lot Subbu -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED]
Re: [ccp4bb] FPLC vs Duo Flow
Thanks to all those who responded. Here is a summary of the responses. Overall, there are mixed feelings, but there is a clear bias towards Akta, with a better service and durability – individual experiences with service might be due to geographical distributions? Filip ___ Original question: Hi, I believe this subject has been touched briefly before, but does anyone have any strong feelings before or against using an Akta FPLC/purifier versus a Biorad Duo Flow? The Biorad Duo instruments are significantly cheaper; are they however also 'as good' as GE Healthcare? I'm especially interested in comments from people who have used both instruments before. Cheers Filip ___ Hi Filip, I use both Biologic Duo Flow and Akta Explorer extensively. In my opinion the Biorad equipment is as good and realiable as the Akta for most routine uses. I generally prefer the Akta Explorer over the Biorad system but its mostly a personal preference. In my opinion, the following are the relative advantages of one system over the other. Advantages of Biorad: 1. It is easy to use and maintain/clean. 2. The software interface is straight forward and user firendly. The Akta software appears to deter new users! 3. The Biorad system is highly modular. So, upgrading the equipment or adding more capabilities is easy and cheap. 4. The system pressure is far lower than Akta at a particular flow rate. Advantages of Akta: 1. The Akta software is far superior in terms of capability (display/data analysis), although not as user friendly in terms of creating a program. 2. The fraction collectors are more robust and reliable, although the Biorad fraction collector seems to have improved. 3. The Akta system is less sensitive to air in the buffer and runs perfectly fine without degassing buffers. It is fairly easy to get air-trapped in the Biorad pump. I would not hesitate to chose the Duo Flow over the basic Akta FPLC if cost is a major concern. Hope this helps, -Anirban - Dear Filip, I would recommend getting a demo from the manufacturer. It's hard to decide based on other people's recommendations. Having said this I also must add that I am an avid fan of the AKTA systems, although their prices are high. Still, a Purifier can be obtained for around 30K, which is comparable with the 20K we have heard for the DuoFlow system with similar capacity. The DuoFlow is overall cheaper, however it also has the distinctly 80-ies feeling of a box-modular system. The fancy names of the components don't help either – for example, why does there have to be a special valve kit to double the performance of the standard 10 and 40 ml/min pumps? Everything has to be configured piece-by-piece and the software is somewhat clumsy, as compared to the sleek Unicow (Unicorn) that runs the AKTA systems. The advantages of the system are essentially the same as its disadvantages – namely that you can configure DuoFlow to be as simple or as complicated as you wish, and more importantly if money is tight – theoretically, additional pieces can be bought later as the money situation improves. The slight problem with this is that you get a system where individual components are ageing at different rates. So, as I said – ask the rep to bring you a system for a few days – they're pretty small so it shouldn't be a serious problem for them, especially if you can hint on trying to decide on it versus AKTA, or on the potential to buy more than one J Good luck, Artem Actually I have to take back the price comment. I cannot find a way to build something for less than 33-35K that'd be comparable to the 34K for AKTA Purifier. Perhaps they quote you different prices, though. Artem - Hi Filip, The support for the Akta machines since they have been bought by GE has been absolutely terrible in our experience. Their engineers know less about the machines than we do and that was not the case before they became part of the giant GE corporation. The Akta that we purchased had defective pumps and it took 6 months for them to acknowledge the problem and they agreed to replace them only when the Howard Hughes purchasing office threatened to take action against the company. So, the bottom line is the machines are well engineered thanks to the great design of the old Amersham engineers, but GE does not care about this market (chump change compared to the other stuff they sell). So as a result the price is high and the service has been terrible in our experience. Just my two cents. Good luck. Antonina Hi, In our lab we have background on both instruments, 2 Biorad and 2 Aktas. We got problems on both, minor ones on Aktas, major ones on Biorads, with expensive fixing on Aktas and cheaper fixings on Biorads, but efficient fixing on Aktas and unsolved
[ccp4bb] FPLC vs Duo Flow
Hi, I believe this subject has been touched briefly before, but does anyone have any strong feelings before or against using an Akta FPLC/purifier versus a Biorad Duo Flow? The Biorad Duo instruments are significantly cheaper; are they however also 'as good' as GE Healthcare? I'm especially interested in comments from people who have used both instruments before. Cheers Filip -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED]
