[ccp4bb] Crystal handling

Hi all. I have small and fragile membrane protein crystals with size 40 micron. They like to break when I try take them out with regular loops for freezing. Is there any special tricks to handle them for data collection? Thank you. Theresa

Re: [ccp4bb] Crystal handling (Summary)

Thank you for all the on and off-board replies. The suggestion is to use meshed loops from Molecular Dimensions or Mitegen. I presume both works well to collect data. Theresa

[ccp4bb] Off-topic-Cleavage with TEV protease

Dear all I want to digest a tagged protein with TEV protease, it has disulfide bridges. Is there any way of doing cleavage without DTT? Thank you. Theresa

[ccp4bb] Off-topic: Site-directed mutagenesis

Dear all I would like to get some opinions on site-directed mutagenesis. What are the current methods available? I know the Quick Change, are there others that work better? Thank you.

Re: [ccp4bb] Off-topic: Site-directed mutagenesis‏ (Summary)

Dear all Thank you for the responses. This is summary of the replies. Quick change method: I believe quick change is the standard method. I have utilized a two step method, where two portions of the gene are amplified within the vector (instead of full vector amplification as in quickchange).

[ccp4bb] Membrane protein purification with HIC

Dear all Can membrane proteins purified with hydrophobic interaction chromatography (HIC) without destroying the protein or the resin? Any type of detergents to avoid? Thank you. Theresa

[ccp4bb] Anisotropic diffraction

Dear crystallographers A very basic question, for anisotropic diffraction, does data truncation with ellipsoidal method change the symmetry? For example, if untruncated data is space group P6, will truncated data index as P622 or P2? Thank you. Theresa

[ccp4bb] Anomalous SAXS

Dear all Is there any interesting aspects of metal proteins that can be used with anomalous SAXS similar to MAD in MX? Can metal distance be measured with time-resolved method (ligand binding and so on)? I knnow examples for materials like nanoparticles but how about proteins? Thank you.

[ccp4bb] Off topic: Complexes cloning

Dear all A off-topic question - is there a practical limit for overexpression of membrane protein complexes (about 200 kDa in total)? This includes chaperones and my target proteins. Can I amplify the gene cluster and simply clone into a plasmid? Thank you.

[ccp4bb] Off-topic: NMR and crystallography

Dear all A question for the cross-trained members of this forum - for small sized proteins, is NMR better than crystallography in terms of data collection (having crystals in the first place) and data processing? How about membrane proteins? I would appreciate replies to the board, instead of

[ccp4bb] Heterogeneity during purification

Dear all I am working on a 30 kDa membrane protein which forms a functional dimer. The protein is His-tagged at N-terminal. In small scale expression screening from whole cells, there is only a single band on Western blot at 30 kDa. But, after purification, additional bands appear at 60 and 120

Re: [ccp4bb] Heterogeneity during purification

Dear Tony Yes, there are four cysteines. Theresa On Tue, 9 Jul 2013 06:26:19 +, Antony Oliver wrote: >Theresa, > >Are there any cysteines in your protein? > >Tony. > >On 9 Jul 2013, at 05:01, Theresa Hsu wrote: > >> Dear all >> >> I am workin

[ccp4bb] Identify type of interaction in crystal structures

Dear all How can we identify the type of interactions, whether electrostatic or covalent, between metals and proteins from the crystal structures? Thank you. Theresa

[ccp4bb] Yeast vector

Dear all Is there a single vector for protein expression that can be used in Pichia, Kluyveromyces and Saccharomyces? They are all of the same family, just different genera. Thank you. Theresa

[ccp4bb] In situ data collection

Dear all Could I get some personal reviews on the CrystalHarp plates? Has anyone used it for actual data collection and structure solution in situ? How does it compare with X-ray 'transparent' plates? Thank you. Theresa

[ccp4bb] Membrane fractionation

Dear crystallographers When we purify membrane proteins, we often fractionate the E. coli cell membrane by ultracentrifugation. Is there a need for this step instead of solubilizing the whole cell with detergents? Thank you. Theresa

[ccp4bb] Room temperature data collection

Dear crystallographers Just out of curiosity, is it possible to collect datasets from crystals at room temperature at synchrotron? Are fast detectors like Pilatus useful for this? Thank you. Theresa

[ccp4bb] Thermofluor assay

Dear all Does anyone has experience with Thermofluor assay to find the substrate transported/binding by a membrane protein? My protein does not have any similar structures and the substrate suggested by sequence analysis is not being transported in proteoliposome. I know ITC is good but I am lo

[ccp4bb] Off topic: Homology modeling

Dear all I am working with a membrane protein without known structure. The closest protein in PDB has 10% sequence identity/25% similarity to my protein. What is the best method and software to do homology modeling while I try to get the crystal? Is the ligand binding site prediction reliable?

[ccp4bb] Off topic: Cloning multi genes/multi cistronic in yeast

Dear all One of my human membrane proteins have been described to interact with additional subunits for its activity. To obtain functional form in yeast (Saccharomyces), I can think of two approach of either cloning all the subunits under one promoter or reconstitute in vitro. For the first op

[ccp4bb] Membrane protein assay

Dear all I want to get the community's opinion regarding transport assay of membrane protein. Is it reliable to use whole cell of E. coli instead of purified protein with appropriate fluorescent indicators to calculate kinetics? The reason is some of my mutants are expressed at low levels not e

[ccp4bb] Off topic: Precast gels

Dear all Would anyone knows of source of cheap precast SDS-page gels? Thank you.

[ccp4bb] Yeast membrane protein extraction

Dear all Would you have a good protocol for membrane protein extract from S. cerevisiae? We don't have French press in our lab. Is the Pierce Mem-PER reagent kit useful for proteins (10-12 transmembrane helices) that will be use for crystallization? Thank you. Theresa

[ccp4bb] GFP membrane protein expression

Dear all Is there any method to check membrane protein overexpression using GFP when the C terminus is in periplasm? My reading so far all mention that for C terminus fusion to work, it has to be cytoplasm. Thank you.

[ccp4bb] Crystallization with antibody

Dear crystallographers Trying to crystallize a membrane protein complex of 100 kDa with a soluble protein of 20 kDa which is interact with the membrane protein. So far, no co-crystals in > 200 conditions. Some conditions gave crystals but mass spec of crystals show only either one protein prese

[ccp4bb] Protein thermostability

Dear all I have two homologues structures, from a mesophilic bacterium and a thermophilic bacterium. Is there any software or server that can calculate the differences that contribute to thermostability, e.g. proportion of amino acids that form Hydrogen bonds and number of prolines? Thank you.

[ccp4bb] 2D and 3D profile in integration

Dear crystallographers What does 2D (Mosflm?) and 3D (XDS?) profile fitting means for data integration? What is the guideline for using either one? References to any literature is highly appreciated. Thank you.

[ccp4bb] Structure alignment

Dear all I have two proteins in PDB each with two subunits. One of the subunits can align well in both. How do I calculate rmsd for the aligned subunits and the other non-align subunits *separately*? Thank you.

Re: [ccp4bb] How to identify unknow heavy atom??

Does EXAFS requires same amount of samples as ICP-MS/ICP-AES? Theresa On Tue, 24 Jul 2012 12:55:31 -0500, Jacob Keller wrote: >Perhaps also exafs should be mentioned--I believe the various ion species, >redox states, and even binding geometry can be determined. > >JPK > > > > >-- >

[ccp4bb] Space group R32 and H32

Dear all I have a confusion on the space group R32 and H32. For a cell parameter of a = b not equal to c, alpha=beta, not equal to gamma, is it considered as R32 or H32? I tried searching the mail list archives but it does not help a beginner crystallographer like me. I also have another basi

[ccp4bb] Changing HETATM to ATOM record

Dear all Is there any tool in CCP4 that can change all HETATM records in PDB file to ATOM? I need the metals to be defined as ATOM for calculation of its normal mode with elNémo. Thank you.

[ccp4bb] Purify non-stable protein

Dear all I made deletion mutation of a stretch of 20 amino acids on my protein. I can purify and crystallize wild type protein but not the mutated. Mass spec on gel separated protein shows degradation of mutant losing about another 150 amino acids. Is there any way of purifying this non-stable

[ccp4bb] Plastic and glass plates

Dear all Is there any pros and cons of using plastic plates for LCP crystallization? The glass is clearer but it is very difficult to open. Thank you.

[ccp4bb] Protein volume

Dear all I have two membrane protein structures. Is there any tool to calculte the volume of transmembrane domain and solublle domain separately for comparison? Thank you.

[ccp4bb] Off topic: Synchrotron

Dear all I have off topic questions. Why can't electrons continuously circulate the ring at synchrotrons instead being refilled every few hours? Does new electrons injected into the ring affect MX data collection? Secondly, is it impossible to have all MX beamlines with insertion device as th

[ccp4bb] Indexing with XDS

Dear all I took some images from test crystal and tried to index them to get cell parameters, not enough for structure solution. I can see spots at 3.5 A with ADXV but when I indexed, XDS reports up to 3.0 A in INCLUDE_RESOLUTION_RANGE in XDS_ASCII.HKL. Questions: 1. How do I know that the sp

[ccp4bb] Reservoir buffer

Dear all In *initial screening* using vapor diffusion crystallization, does it matter whether the reservoir buffer is also the precipitant in the drop or just a high salt solution like 5 M NaCl? Thank you. Theresa

Re: [ccp4bb] refining against weak data and Table I stats

Being a beginner crystallographer, may I ask a basic question? On how many occasions does it make a *biological* difference between having a structure at 1.42 and 1.6 A? I think this question also extends to adding in water molecules just to make statistics look good. Thank you. Theresa On T

[ccp4bb] Membrane protein overexpression

Dear all I have a general question on membrane protein overexpression in E. coli. Are E. coli proteins expressed in E. coli always better in terms of yield, lipid preservation and so on than another homologous protein? I am aware that it is different for *crystallization* because of flexible lo

[ccp4bb] Molecular cloning software for linux

Dear all Is there any good molecular cloning software for linux? It should read plasmid constructs in Genbank format, do assembly of plasmid sequencing results, display the sequencing trace and highlight regions where there are mutations based on relative QV values, has a list of built-in restr

[ccp4bb] Cell free expression

Dear all Out of curiosity, are cell free expression systems using E. coli or insect cell extracts useful for membrane protein expression-crystallization? What are the costs associated with these systems? Thank you. Theresa

[ccp4bb] Off topic: Oligonucleotide purity

Dear all What is the right (cost wise) purity level for DNA oligonucleotide synthesized for cloning, site-directed mutagenesis and protein/DNA co-crystallisation? Thank you. Theresa

[ccp4bb] Philosophical question

Dear all I have a somewhat philosophical question. Why do all protein sequences start with a methionine (not referring to mature/processed form)? What is so special about methionine and cannot be replaced by other amino acids? Second, how does the ribosome know the first start codon is for meth

[ccp4bb] Oligomerization state

Dear all I have a His-tagged membrane protein with unknown oligomerization state. But I am worried that tag addition may induce different state than in native and affect its crystallizability. Is there a single method that can determine the oligomerization state with nearly 100% accuracy? I ha

Re: [ccp4bb] Off topic: Oligonucleotide purity (Summary)

Dear all I received off-board replies on this subject. Most people use standard desalting for all cloning and mutagenesis experiments regardless of length. One reply recommended desalting for up to 40 bases and HPLC for longer oligos because of larger ratio of (n-1) contaminants. For crystalliz

[ccp4bb] Comparison of membrane protein expression

Dear all I want to try a factorial approach for my His-tagged membrane proteins. The screening parameters are E. coli strains, temperature and media. Is there any simple but representative method I can use to compare the expression level without isolating the membrane? Is whole cell lysis and W

[ccp4bb] PILATUS data collection

Dear crystallographers Is there a good source/review/software to obtain tips for good data collection strategy using PILATUS detectors at synchrotron? Do we need to collect sweeps of high and low resolution data separately? For anomalous phasing (MAD), does the order of wavelengths used affect