CCP4 Bulletin Board Archive

Sunday, 4 September 2011

Hiro Tsuruta

From: Tsutomu Matsui
Date: 28 August 2011 11:24

It is with great sadness that we announce that Dr. Hirotsugu (Hiro) Tsuruta, a senior scientist and biophysicist in the Structural Molecular Biology program at Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC, passed away on August 25, 2011, in Japan, after a courageous fight with cancer. Funeral services took place in Sasebo, Japan, on August 27.

Hiro worked at SSRL for more than 20 years, and with great dedication and scientific insight provided leadership for the build-up of the SSRL biological small angle x-ray scattering research program, and a bioSAXS beam line facility that today serves a large user community. He also served the international community in many ways. His contributions were numerous, and he leaves an international legacy in this growing area of science, for which he pursued new developments until the end.

SSRL will organize an event to honor his contributions, and information will follow when plans become available.

Britt Hedman and Keith Hodgson

Rigaku MicroMax-007 system available

From: Erin Curry
Date: 26 August 2011 18:53

Hi everyone,
Due to recent downsizing, a complete Rigaku MicroMax-007 system needs
a new home! R-Axis IV++ detector, computer, software and all other
components included. Please contact me off-list for details.
Happy Friday!
Erin

crystallization of cytochrome C from horse heart

From: Danilo Belviso
Date: 26 August 2011 15:19

Dear all,

I would like to crystallize cytochrome C from horse heart, to perform a soaking. I have tried the crystallization conditions that are reported by many papers (1) but I have not obtained cytochrome C crystals until now.
Only big objects (800micrometer) that do not seem single
crystals (in attachment_1) are been obtained (conditions are in attachment_2).
Conditions were performed using cytC from horse heart of Sigma Aldrich and with protein obtained in our lab. The result was the same.

May someone give me some tricks to crystallize cyt C?

Thanks a lot.

Danilo

(1) - High res 3D structure of Horse Heart Cytochrome c J. Mol.Biol.
(1990), 214,585-595
- The low ionic strength crystal structure of horse cytochrome c at 2.1A
resolution and comparison with its high ionic strength counterpart
Structre (1995), 3(7)
- High resolution X-Ray crystallographic structure of bovine heart
cytochrome c and its application to the design of an electron transer
biosensor
Proteins (2008), 70, 83-92

Crystallization trials for CytC apo from Horse Heart

Trials with low ionic strange
Conditions	Drop 4μL	2μL ([CytC]=25mg/ml in [Na3PO4]=50mM pH7) + 2μL (PEG1000 25%w/v in [Na3PO4]=50mM pH7)	Drop: We put on cover slip 2μL of solution B and after we added 2μL of solution C
	Reservoir 700μL	PEG1000 30%w/v in [Na3PO4]=50mM pH7	The reservoir was made of: 250μL of solution A 600μL of PEG1000 50% (HR2-523 Hampton) 150μL of milliQ water
	Method	Hanging drop (Nextal Plate)
	Temperature	20°C

Solution A (buffer phosphate 200mM pH7): the solution was made of trisodic phosphate (71908 Fluka) titrated with NaOH (480507 Carlo Erba) and HCl (403912 Carlo Erba) to obtain pH 7.

Solution B (buffer phosphate 50mM pH7, PEG1000 25%w/v): the solution was made of 25μL of solution A, 50μL of PEG 1000 50%w/v (HR2-532 Hampton) and 25μL of milliQ water.

Solution C ([CytC]=25mg/ml, buffer phosphate 50mM pH7,PEG1000 25%w/v): the solution was made of 10mg of CytC (105201 Sigma Aldrich) stabilized in 100μL of solution A. The protein solution was diluted with 300μL of milliQ water

identifying hinges in flexible proteins

From: Stephen Cusack
Date: 26 August 2011 10:25

We have structures of two states of a large multi-domain protein in which domain
movements of up to 30 degrees are observed. Is there a programme available which
attempts to determine rigid-body units and hinge regions in an 'unbiased' way?
thanks for your suggestions,
Stephen Cusack

----------
From: Matthew BOWLER

Dear Stephen,
I find DynDom very good (http://fizz.cmp.uea.ac.uk/dyndom/) otherwise the morph server (http://molmovdb.mbb.yale.edu/molmovdb/morph/) also performs similar analysis, cheers, Matt. Matthew Bowler

----------
From: Martyn Winn

DynDom version 1.5 is part of the CCP4 suite.
There is an interface for it under Program List.

Although Steve Hayward appears to have a new version DynDom3D which we
don't have. No idea if it would be better for your case.

HTH
Martyn

co-crystallization

Hi ,

I am co-crystallizing a protein with compound and would like to know how much of compound to add to protein solution to start with. I know that the protein binds compound in a 1 to 1 ratio but also noticed that the compound precipitates out of solution when DMSO is diluted off. Where should I start of? A 1 protein :2 compound ratio or more? And what is the best method to determine if the binding is homogeneous (that all protein has got a compound in it)?

Any suggestions would help. Thanks

----------
From: Shilong Fan

for me, I prefer to sock these compounds into your crystal. it will much more easy than co-crystallizaiton. But each protein should be different.

Normally when I start co-crystallization with small compound, I will set up the complex with 1:1.2 molar ratio as first trial to see what should happen.

As DMESO, you can't get rid of them. But you can find as much lower concentraiton as you can.

----------
From: Annie Hassell

Hi YT—

We normally prepare our ligand stocks in DMSO and add this to the protein in 3-fold molar excess. The majority of our ligands are quite insoluble and precipitate when the DMSO concentration decreases upon addition to the protein……. so I am not surprised that you are seeing this. If your compound does not bind your protein tightly, you might consider using a 5-fold molar excess of ligand.

Some proteins crash out if the protein concentration in high when you add the ligand. For those situations, we complex the ligand with dilute protein (1-2 mg/ml), and then concentrate this for crystallization trials. I have had proteins where we had to complex the dilute protein with ligand, and then let it sit overnight at 4C before we concentrated the protein. We normally incubate the protein+ligand at 4C for 1-3 hours for binding before we set up the crystallization experiments.

Another scenario might be addition of ligand to the protein followed by incubation at room temp for ~1hr. Then centrifuge at 4C, keep protein at 4C and set up your trays.

Hope this helps!

annie

----------
From: Prince, D Bryan
Date: 25 August 2011 14:38

Dear TY,

Typically between 5-10x molar concentration over the protein is enough to ensure binding when the IC50 is uM to low mM. For tighter binding compounds (nM to low uM), 2-5x is sufficient. Whatever you do, when the precipitate occurs DO NOT REMOVE it. I learned to my chagrin that you change all the dynamics of the drop when you do. I ended up with empty crystals until I left the precipitate in place. Think of it this way—

Free protein + compound ↔ protein:compound complex + precipitate (mix of protein + compound)

If you change the equilibrium by removing the precipitate, you remove the "pressure" on the P:C complex, and it will dissociate to P + C. The precipitate acts as a reserve of protein and compound, thus favoring (or stabilizing) the P:C complex. I set drops up as a slurry frequently, and if I get crystals, they always have the compound bound. Pay attention to the drops if you are screening, because it will be important to note what makes the precipitated solution better (clear drops=solubilizing) or worse (aggregated drops=decreased solubility of your complex). You can also try suspending your compound in LMW PEG's (200-400 FW) instead of DMSO. Either way, try using DMSO (~20%) or LMW PEG (~30%) depending on your crystallization conditions as a cryoprotectant. Any crystals that grow have some small amount of those agents in them already, so they should be more tolerant of them in higher concentrations.

Best of luck!

Bryan

----------
From: Roger Rowlett

Successful complexation depends on the concentration of protein, ligand, and the Kd of the protein-ligand complex. For Kd>>[protein], you will probably require [ligand] > 10 x Kd. As Kd approaches [protein], slightly superstoichiometric quantities will be sufficient for full occupancy. For Kd < [protein], stoichiometric quantities of ligand will suffice. Basically you need a [ligand] that puts near saturation on the binding isotherm.

Cheers.

--

Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

----------
From: <Herman.Schreuder

If you do the calculations, you will find that you need a FREE ligand concentration of >10 * Kd to get >90% occupancy of the binding site.

If you have e.g. a ligand with a Kd of 100 nM, you would need a free ligand concentration of 1 µM. However, a solution of 10 mg/ml of a protein of 30 kDa, has a protein concentration of 333 µM, so in theory you should have a total ligand concentration (free + bound) of 334 µM. In pratice, some of the ligand may have been degraded during (prolonged) storaged, or the compound may not be as pure as the chemist would have wished it to be, so it is wise to use a safety margin of at least two. We normally use 1-2 mM compound to be on the safe side. Having too much ligand usually does not hurt, except that you use more compound, but with too little ligand you end up with an empty binding site and you will have to repeat the experiment with more ligand.

Best,

Herman

Saturday, 3 September 2011

Web Seminar: An Insider's Guide to CrystalClear and d*TREK: Improve Your Results with these New Features and Tips

From: Angela Criswell

Dear colleagues,

I would like to draw your attention to an upcoming free, educational webinar to be presented by Jim Pflugrath, Ph. D. titled "An Insider's Guide to CrystalClear and d*TREK: Improve Your Results with these New Features and Tips". In this webinar, Jim will discuss three important new features or tips for using CrystalClear and d*TREK which will lead to better results: lower Rmeas and higher <I/sigmaI>. New input parameters for finding spots is one new feature. Another feature is a better way perform positional refinement before and during Bragg reflection integration. A third tip is how to improve your scaling results by changing the input parameters to dtscaleaverage. These tips and others will give you better results with CrystalClear.

This webinar is scheduled to occur on Thursday, September 1st 10:00 AM CDT (8:00 AM PDT / 4:00 PM GMT). You can find more information, including a registration link at: http://www.rigaku.com/protein/webinars.html.

Best regards,
Angela Criswell

--
Angela R. Criswell, Ph. D.
VP, Life Sciences
Rigaku Americas Corporation
9009 New Trails Drive
The Woodlands, TX 77381 USA
URL: http://www.rigaku.com

2nd MX User Workshop at Diamond - September 7th-8th, 2011

From: Thomas Sorensen

Dear All

I quick reminder - last chance to sign up for the 2nd MX User Workshop at Diamond - the workshop is part of the Synchrotron User Meeting, September 7th-8th, 2011. Workshop sessions include presentations from staff and users on latest developments, spectroscopy, crystal dehydration and room temperature data collection, using microfocus beam and how to get the most from your beamtime.

Workshop info: http://www.diamond.ac.uk/Home/Events/Synchrotron_User_Meeting_2011/MX.html
Registration: http://www.diamond.ac.uk/Home/Events/Synchrotron_User_Meeting_2011/Registration-system.html

Best wishes

Thomas Sorensen
Principal Beamline Scientist
Macromolecular Crystallography
Diamond Light Source

loop building with ARP/wARP

From: Gregory Bowman
Date: 25 August 2011 14:12

Hi all,

I have a large disordered loop (33aa) for a 2.0 Å dataset for which the rest of the structure is well-defined, and phases are decent (Rwork=19.6, Rfree=24.6). I can see some broken up density at one end, but have been unable to convincingly build into into this region manually. I would like to try arp/warp to improve density or possibly extend the loop termini but have been thwarted by some technical problems and would appreciate any advice. I've installed ccp4 6.2.0 on Mac OS X (10.6.8) with fink, both as 32-bit and 64-bit to allow me to run ARP/wARP (version 7.1) from the ccp4i GUI. I've been unable to get "loopy" to work, and also the standard ARP/wARP classic.

When I run "ARP/wARP Loops" (a.k.a. loopy), I get the message in the log file: "Couldn't find any loop to build, loopy will simple copy the pdb file". The program is able to read the sequence .pir file and match it to the structure, and seems from the sequence alignment to identify the regions of the sequence for which there is no structure. I'm guessing that I'm not putting some obvious parameter into the GUI/starting script, but I don't know what that might be. The script generated from the GUI (21_loopy.def) is pasted below.

When I try to run ARP/wARP classic for loop building, I get the following message in the logfile:

QUITTING ... ARP/wARP module stopped with an error message:

REFMAC

*** Look for error message in the file:

/Users/gbowman/Greg/20_warpNtrace_refine.last.log

At the end of the warpNtrace_refine.last.log file, the program reports what seem to be close contacts. Since I don't see this in the structure, and don't have any problems or errors like this when I refine the structure with REMAC, is this from rebuilding, and is this the problem that halts REFMAC?

Thanks!

Greg

========= ========= ========= ========= ========= =========

20_warpNtrace_refine.last.log

========= ========= ========= ========= ========= =========

<snip>

Input file :restraints.pdb

NUMBER OF MONOMERS IN THE LIBRARY : 11465

with complete description : 11465

NUMBER OF MODIFICATIONS : 53

NUMBER OF LINKS : 66

I am reading libraries. Please wait.

- energy parameters

- monomer"s description (links & mod )

</FONT></B>

Number of atoms : 2463

Number of residues : 859

Number of chains : 3

I am reading library. Please wait.

mon_lib.cif

INFO: link is found (not be used) dist= 1.907 ideal_dist= 1.432

ch:AA res: 8 GLY at:CA .->ch:AA res: 50 TYR at:OH .

INFO: link is found (not be used) dist= 2.126 ideal_dist= 1.645

ch:AA res: 13 ARG at:NH1 .->ch:AA res: 54 MET at:SD .

INFO: link is found (not be used) dist= 1.820 ideal_dist= 1.462

ch:AA res: 13 ARG at:NH1 .->ch:AA res: 54 MET at:CE .

INFO: link is found (not be used) dist= 1.757 ideal_dist= 1.395

ch:AA res: 13 ARG at:NE .->ch:AA res: 119 PHE at:CD1 .

INFO: link is found (not be used) dist= 1.572 ideal_dist= 1.462

ch:AA res: 13 ARG at:NH2 .->ch:AA res: 125 LEU at:CD2 .

INFO: link is found (not be used) dist= 1.751 ideal_dist= 1.513

ch:AA res: 33 ILE at:CG2 .->ch:AA res: 39 PRO at:CG .

INFO: link is found (not be used) dist= 1.451 ideal_dist= 1.513

ch:AA res: 33 ILE at:CG2 .->ch:AA res: 39 PRO at:CD .

INFO: link is found (not be used) dist= 1.759 ideal_dist= 1.524

ch:AA res: 58 LYS at:CD .->ch:AA res: 130 LEU at:CB .

INFO: link is found (not be used) dist= 1.725 ideal_dist= 1.513

ch:AA res: 58 LYS at:CD .->ch:AA res: 130 LEU at:CD1 .

INFO: link is found (not be used) dist= 1.578 ideal_dist= 1.524

ch:AA res: 83 ARG at:CD .->ch:AA res: 101 PRO at:CG .

INFO: link is found (not be used) dist= 1.843 ideal_dist= 1.457

ch:AA res: 83 ARG at:NE .->ch:AA res: 101 PRO at:CB .

INFO: link is found (not be used) dist= 0.666 ideal_dist= 1.457

ch:AA res: 83 ARG at:NE .->ch:AA res: 101 PRO at:CG .

INFO: link is found (not be used) dist= 1.275 ideal_dist= 1.510

ch:AA res: 83 ARG at:CZ .->ch:AA res: 101 PRO at:CB .

INFO: link is found (not be used) dist= 1.102 ideal_dist= 1.510

ch:AA res: 83 ARG at:CZ .->ch:AA res: 101 PRO at:CG .

INFO: link is found (not be used) dist= 1.191 ideal_dist= 1.457

ch:AA res: 83 ARG at:NH1 .->ch:AA res: 101 PRO at:CA .

INFO: link is found (not be used) dist= 0.690 ideal_dist= 1.457

ch:AA res: 83 ARG at:NH1 .->ch:AA res: 101 PRO at:CB .

INFO: link is found (not be used) dist= 1.891 ideal_dist= 1.457

ch:AA res: 83 ARG at:NH1 .->ch:AA res: 101 PRO at:CG .

INFO: link is found (not be used) dist= 1.352 ideal_dist= 1.457

ch:AA res: 165 VAL at:CG1 .->ch:AA res: 172 TRP at:CZ3 .

INFO: link is found (not be used) dist= 1.440 ideal_dist= 1.457

ch:AA res: 165 VAL at:CG1 .->ch:AA res: 172 TRP at:CE3 .

INFO: link is found (not be used) dist= 1.367 ideal_dist= 1.320

ch:AA res: 172 TRP at:CD1 .->ch:AA res: 176 GLU at:OE1 .

INFO: link is found (not be used) dist= 1.447 ideal_dist= 1.275

ch:AA res: 172 TRP at:NE1 .->ch:AA res: 176 GLU at:OE1 .

INFO: link is found (not be used) dist= 1.760 ideal_dist= 1.360

ch:AA res: 172 TRP at:CH2 .->ch:AA res: 253 ARG at:CZ .

INFO: link is found (not be used) dist= 1.690 ideal_dist= 1.309

ch:AA res: 187 TYR at:OH .->ch:AA res: 198 ASP at:OD2 .

--------------------------------

--- title of input coord file ---

PDB_code:xxxx

PDB_name:----

PDB_date:XX-XXX-XX

--------------------------------

Number of chains : 3

Total number of monomers : 859

Number of atoms : 2463

Number of missing atoms : 0

Number of rebuilt atoms : 0

Number of unknown atoms : 0

Number of deleted atoms : 0

Number of bonds restraints : 1863

Number of angles restraints : 2492

Number of torsions restraints : 1286

Number of chiralities : 266

Number of planar groups : 318

loop_

_atom_type_symbol

_atom_type_scat_Cromer_Mann_a1

_atom_type_scat_Cromer_Mann_b1

_atom_type_scat_Cromer_Mann_a2

_atom_type_scat_Cromer_Mann_b2

_atom_type_scat_Cromer_Mann_a3

_atom_type_scat_Cromer_Mann_b3

_atom_type_scat_Cromer_Mann_a4

_atom_type_scat_Cromer_Mann_b4

_atom_type_scat_Cromer_Mann_c

N 12.2126 0.0057 3.1322 9.8933 2.0125 28.9975 1.1663 0.5826 -11.5290

C 2.3100 20.8439 1.0200 10.2075 1.5886 0.5687 0.8650 51.6512 0.2156

S 6.9053 1.4679 5.2034 22.2151 1.4379 0.2536 1.5863 56.1720 1.0496

O 3.0485 13.2771 2.2868 5.7011 1.5463 0.3239 0.8670 32.9089 0.2508

========= ========= ========= ========= ========= =========

21_loopy.def

========= ========= ========= ========= ========= =========

#CCP4I VERSION CCP4Interface 2.1.0
#CCP4I SCRIPT DEF loopy
#CCP4I DATE 25 Aug 2011 08:17:34
#CCP4I USER gbowman
#CCP4I JOB_ID 21
#CCP4I PROJECT GDB210i
#CCP4I TASKNAME loopy
#CCP4I LOG_FILE 21_loopy.log
#CCP4I EDIT_SCRIPT 0
#CCP4I HTML_LOG 0
#CCP4I REMOTE 0
#CCP4I SERVER_HOST localhost
#CCP4I SERVER_PORT 4441
#CCP4I DATABASE_SERVER 0
#CCP4I DATABASE_SERVER_HOST localhost
#CCP4I DATABASE_SERVER_PORT 0

ABORT_LEVEL 8
ATOM_RADIUS 0.74
B_FACTOR 20
B_FACTOR_SIDE_CHAIN 26
CA_DISTANCE 3.8
CA_DISTANCE_ERROR 0.3
CELL_1 38.670
CELL_2 61.426
CELL_3 76.771
CELL_4 90.00
CELL_5 104.17
CELL_6 90.00
CHECK_FIRST_ANGLE 1
C_ANCHOR "A 1213"
DIR_EXT_MAP_FILENAME TEMPORARY
DIR_LOOPFIT_EXE_FILENAME "Full path.."
DIR_LOOPFIT_LOG_FILENAME TEMPORARY
DIR_MAP_FILENAME GDB210i
DIR_MAP_FROM_MTZ TEMPORARY
DIR_MESSAGE_FILENAME GDB210i
DIR_MTZ_FILENAME GDB210i
DIR_OUTPUT_PDB GDB210i
DIR_PDB_INPUT_FILENAME GDB210i
DIR_PRELOOPFIT_PDB TEMPORARY
DIR_SAVE_LOOP_NAME GDB210i
DIR_SAVE_PROP_PDB GDB210i
DIR_SEQIN,0 ""
DIR_SEQIN,1 GDB210i
DIR_STRUCTURE_TO_C "Full path.."
DIR_STRUCTURE_TO_N "Full path.."
DIR_XML_MESSAGE_FILENAME GDB210i
DUMMY_REMOVAL_THRESHOLD 0.1
EXTEND_GAP_SMALLER_THAN 5
EXTEND_REFINEMENT 0
EXT_MAP_FILENAME ""
F1 FWT
FITTARGET 1
FORCE_MIN_CAS_KEPT 1
GRID_NUMBER 378
GRID_TYPE 0
INCLUDE_ALL 1
INCLUDE_CHAINS ""
KEEP_NEG_DENS_HALFWAY 0
LIKELIHOOD_THRESHOLD -5

LOOPFIT_EXE_FILENAME /Users/gbowman/Greg/arp_warp_7.1/bin/bin-i386-Darwin/loopfit

LOOPFIT_LOG_FILENAME loopfit.log

LOOPS_TO_BUILD A1213(33)A1245

LOOP_BOTH_WAYS 1

LOOP_DENSITY_CUTOFF_NO 100

LOOP_LENGTH 33

LOOP_MAIN_CHAIN_DENS_NO 12

LOOP_OVERLAP 0

LOOP_RMS 0.5

LOOP_SEQUENCE ""

LOOP_STRUCTURE_CUTOFF_NO -1

LOOP_STRUCTURE_MIN_NO 0

LOOP_STRUCTURE_THRESHOLD -7

LOOP_TO_C 1

MAP_FILENAME ""

MAP_FROM_MTZ 160BDNAmerged2011aug09_loopy2.map

MAP_INPUT_MODE MTZ

MAX_DISTANCE_BETWEEN_ANCHORS -1.

MAX_LOOPS_AFTER_MC_PLANE 50

MAX_LOOP_LENGTH 15

MAX_NO_CAS_KEPT 5

MESSAGE_FILENAME loopy.msg

MESSAGE_LEVEL 5

MET_IS_SEL,0 0

MET_IS_SEL,1 0

MINIMAL_DISTANCE 0.45

MODE_LOOPY 0

MTZ_FILENAME 160BDNAmerged2011aug09_refmacGDB210i-215.mtz

NMOL,0 1

NMOL,1 1

NRES,0 ""

NRES,1 272

NRESMOL,0 ""

NRESMOL,1 272

NSEQFILES 1

N_ANCHOR "A 1245"

OUTPUT_PDB 2011aug24-GDB210i-renameDNA-3_loopy1.pdb

OVERLAP_REMOVAL_THRESHOLD 0.1

PDB_INPUT_FILENAME 2011aug24-GDB210i-renameDNA-3_refmac1b.pdb

PHI PHWT

PRELOOPFIT_PDB pre_loopfit.pdb

REFINEMENT 1

REMOVAL_FACTOR 1.

SAVE_BEST_NUMBER 2

SAVE_LOOPS 1

SAVE_LOOP_BASENAME 2011aug24-GDB210i-renameDNA-3_loop

SAVE_LOOP_DIR /Users/gbowman/Greg

SAVE_LOOP_NAME 2011aug24-GDB210i-renameDNA-3_loop1.pdb

SAVE_LOOP_PROP_BASE 2011aug24-GDB210i-renameDNA-3_proposed

SAVE_LOOP_PROP_DIR /Users/gbowman/Greg

SAVE_PROP_PDB 2011aug24-GDB210i-renameDNA-3_proposed1.pdb

SEQIN,0 ""

SEQIN,1 160B.pir

SHELL_THICKNESS 1

SHOW_DETAILS_DENSITY 0

SHOW_DETAILS_LIKELIHOOD 0

SHOW_PRUNING_DETAILS 0

SPACEGROUP P21

SPACEGROUP_NUMBER 4

STRUCTURE_TO_C /Users/gbowman/Greg/arp_warp_7.1/bin/bin-i386-Darwin/set_1200_2C_my_0.3b_cos_log.llh

STRUCTURE_TO_N /Users/gbowman/Greg/arp_warp_7.1/bin/bin-i386-Darwin/set_1200_2N_my_0.3b_cos_log.llh

TITLE "build 1213 loop with loopy"

USER_SETTING_MAX_NO_CAS 0

USE_PIR_FILE 1

WEIGHT_DENSITY 1.

WEIGHT_DISTANCE 1.

WEIGHT_STRUCTURE 1.

XML_MESSAGE_FILENAME loopy.xml

========= ========= ========= ========= =========

----------
From: Victor Lamzin

Dear Greg,

Try ARP/wARP 7.2, released a few days ago, which is better, more stable and should have improved flash of error messages to the log files.

ARP/wARP Loopy will not build a 33-residue loop at once, but Classic model building may get the gap shortened.

Go to www.arp-warp.org for download. In parallel, from the same page you can submit Classic protein model building for remote execution in Hamburg, which also has ARP/wARP 7.2 installed.

Please get back if problems remain.

With best regards,
Victor

Aging PEGs

From: Jacob Keller
Date: 24 August 2011 20:18

A while ago I measured the pH's of old and new PEGs and found them
very different, and internally attributed all "old vs new PEG issues"
to pH. Upon reflection, this seems too simplistic. Are there other
known mechanisms of crystallization capacities of PEGs of various
ages?

Jacob Keller

*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
*******************************************

----------
From: Jim Pflugrath

PEGs have small amounts of anti-oxidants added to them by the manufacturer.
I think different manufacturers may use different compounds.

And you might imagine that PEGs themselves with their -OH groups go from
alcohol to adelhyde to carboxylate.

----------
From: Craig A. Bingman

Commercial polyethylene glycol is contaminated with polymers that have aldehyde groups at the ends. Other impurities include some amount of internal epoxide linkages. The aldehyde groups can be additionally oxidized to carboxylic acids. I would assume that oxidation to terminal carboxylic acids explains the change in PEG pH vs. time. The pH will also change as the PEG solution comes to equilibrium with atmospheric carbon dioxide. The aldehyde content increases with time after the PEG is dissolved in water and stored under normal atmospheric gas. These PEGs can react with protein amine groups.

----------
From: <mjvdwoerd

PEG is a polymer and it can be made by anionic or cationic polymerization. Whichever you use, you go "the other way" to terminate the reaction at an appropriate time (so you have the molecular weight you want). So when you start with an acid, you terminate with a base and vice versa. If you terminate with a base, your final pH is going to be high (presumably > 7) and if you terminate with an acid, the pH is going to low.

It is therefore important to keep track of your lot number (because as long as the lot number is the same, the treatment was the same). For crystallization recipes that do not involve buffers (there are some!) this is essential, because PEG and PEG are not the same thing (and you should always pH the solution before you use it, so you have a reference point - remember we do not know what we have in our crystallization drops, but we do know what we put into them to make them). Even if the PEG was made by the same process, the manufacturer is concerned with stopping the polymerization at the right time, but not "how hard" they stop it. In other words, the solution might be pH 8 or pH 11 when it is done.

So when you say that you measured different PEGs and found the pH to be different, that might be accounted for by the way the PEG was made and they may always have been different, irrespective of age.

It is probably not known if "acid PEG" vs "basic PEG" ages at a different speed and with a different mechanism. As you know, the first thing to observe is whether PEG solutions are clear ("water") or slightly colored ("dilute lemonade" :-) because this is a sign of aging. Remember that PEGMME is much more sensitive to aging than PEG itself (you should store MME solutions in the dark). And if the PEG is stored in dry form, it is not very easy to age it by chemical reaction, because nothing is "swimming", but "not easy" is not the same as "impossible".

My 2 cents worth.

Mark

----------
From: Prince, D Bryan

For those of us truly controlling types :), I used to make the PEG
solutions and filter them over a Bio-Rad resin that filtered out all the
junk added to stabilize the PEG solution. Then, of course I had to
freeze all my PEG solutions in aliquots, or wrap them in foil and store
at 4C in the dark. This would take several days, depending on the FW of
the PEG. If you are really sensitive about what is in your PEG
solutions, try GC-grade PEG's. The FW profile is much more restricted
around the reported value (i.e. PEG 3350 molecular biology grade has a
broad peak centered on Mr=3350. PEG 3350 GC-grade has a much tighter
peak profile.) Back before you could buy Crystal Screen I, II or HT, you
had to make the stock solutions, then make the screen. But at least when
you did that, you had all the stocks. Now, I just buy pre-made
solutions, and keep them in a drawer with a date opened written on the
bottle. Isn't progress grand? :)

Bryan

----------
From: Frank von Delft

And now, .... does anybody know of systematic data indicating how consistently all this matters?
phx

----------
From: Van Den Berg, Bert

Is there anything that consistently matters in crystallography? ;-)

----------
From: R. M. Garavito

Time to start digging in the archives. Try looking at work by Fran Jurnak in 1986 (J. Cryst. Growth 76, 577) and Bill Ray's work in 1985 (Analytical Biochem 146, 307), and then the works that cite them. I thought this was common knowledge, but I guess it goes in phases.

Aging of poly(oligo)-oxyethylene-based compounds is well known in the surfactant field as it changes the chemical properties of common detergents (Brij, Triton, C10E6, etc.), not only by adding aldehydes and carboxylates to the system, but also by increasing metal binding. It is a sobering sight to see old PEG cross-link your 3-month old crystal: the damn thing wouldn't dissolve after 2 hours in plain buffer, but when I poked it with a glass fiber, the protein oozed out like the center of a cherry cordial, leaving a sad looking deflated shell of a crystal.

Cheers,

Michael

****************************************************************

R. Michael Garavito, Ph.D.

Professor of Biochemistry & Molecular Biology

513 Biochemistry Bldg.

Michigan State University

East Lansing, MI 48824-1319

From: Hargreaves, David
Date: 5 September 2011 14:43

I went through the peg solid stocks in our cupboard and noticed some of them smelled acrid. I probably won’t use them. Not sure whether they’re chemically decomposing. Maybe fossilising?

Dave

David Hargreaves

Associate Principal Scientist

_____________________________________________________________________

AstraZeneca

Merge chains in coot

From: jab
Date: 25 August 2011 15:09

Hi

I've tried to find where to merge chains in coot, eg merge built co-factor
to protein chain, or
merge all my ions (Na, Cl, Mg etc) together.

Any pointers?

Thanks

Jim Brannigan

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From: <Herman.Schreuder
Date: 25 August 2011 15:23

Calculate - Merge Molecules

Herman

Creating CIF Files and Renaming Ligands

From: Ai Fen Chai
Date: 25 August 2011 03:36

Hi all,

I want to add 2 new ligands (that do not exist in PDB) to my structure. I generated the ligands from PRODRG. The problem is that both of the ligands have the same name DRG. After renaming the ligands in both PDB and CIF files, I always get this error message "CIF dictionary does not contain restraints" whenever I try to do refinement. Prior to renaming, both ligands were recognized by COOT when they were named DRG. So, the CIF files themselves are ok and it must be something to do with the renaming that is causing the error. I am wondering whether renaming the ligands has changed the CIF file subtly and causing COOT and other refinement programs to not read it right?

Cheers,

Ai Fen

----------
From: Nicholas Keep
Date: 25 August 2011 09:45

Did you rename the ligand in the CIF file as well? It would be nice if the Prodrg interface allowed you to type in the name of the ligand you want in the CIF and PDB file. Hopefully a global replace in your favourite text editor of DRG with LIG or whatever you want to call your ligand would work. You would have to do that on the separate pdb and CIF files and then merge the two or more CIF files into one if you have more than one ligand. I must admit to this being theory I have not tested it in practice.
Best wishes
Nick
--

Prof Nicholas H. Keep
Executive Dean of School of Science
Professor of Biomolecular Science
Crystallography, Institute for Structural and Molecular Biology,
Department of Biological Sciences
Birkbeck, University of London,
Malet Street,
Bloomsbury
LONDON
WC1E 7HX

----------
From: Ai Fen Chai

Hi all,

Thanks for the suggestions! Anyway, the problems have been fixed! When I edited the CIF files in a mac editor, the file formatting got changed, so a line feed became a carriage return and Coot does not like this as to it they appear to be different files.

The CIF files were fixed by using gedit on the old files on linux machine and
global replace with match case/whole word only option on. They now are recognized by COOT.

Ai Fen

Melting point and heat of fusion

From: Kristof Van Hecke
Date: 25 August 2011 10:06
Subject: [ccp4bb] Melting point and heat of fusion

Dear,

We're wondering what tools there are available in order to estimate the 'melting point' and 'heat of fusion' from a known crystal structure (e.g. coordinate file)..?
It concerns mostly small molecules (> 100 atoms).

Many thanks

Kristof
-----------------------------------
Kristof Van Hecke, PhD
Biomolecular Architecture
Celestijnenlaan 200F
3001 Heverlee (Leuven)
Belgium
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