Welcome to the SCSB Macromolecular X-ray Laboratory |
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The mission of the Sealy Center for Structural Biology and Molecular Biophysics X-ray Crystallography Laboratory is to provide access to state of the art X-ray diffraction instrumentation and the associated support facilities for Center crystallographers.
The Center has recently installed new x-ray diffraction and solution scattering instrumentation for the lab. The new equipment starts with the Rigaku Ultimate Home Lab® which comprises the unsurpassed high-brilliance FR-E++DW Superbright x-ray generator, with the industry standard RAXIS-IV++ crystallography system with both Cu and Cr optics. In addition, we are acquiring the innovative Rigaku BioSAXS-1000, the first SAXS instrument dedicated to biological research in the southern mid-western states, once again marking UTMB as a leader in structural biology resources.
The SCSB X-ray Crystallography resources currently consists of two x-ray area detector systems. The first area detector is a Rigaku R-AXIS-IV++ dual 30cm Imaging Plate detector mounted on the Rigaku FR-E++DW Superbright x-ray generator. The second detector is a Bruker SMART 2K CCD, which can reach upto 0.84 Å resolution. For enhanced data we have available a choice of sample cooling systems. Both systems are equipped with a Cryo Industries of America CRYOCOOLER. A 4 °C Cold Air refrigerated cooling system is available for samples which cannot be frozen, or do not require freezing.
The Rigaku BioSAXS-1000 2D-Kratky camera is mounted on the left port of our Rigaku FR-E++DW Superbright x-ray generator.
This camera offers unsurpassed solution scattering data for macromolecular samples. For more information visit our
SAXNS site.
Instrument scheduling, for qualified users, is available online.
| These are a collection of documents from the SCSB X-ray Crystallography Center and
our research groups. | ||
| X-RAY Policy | ||
| R-AXIS-IV Guide | BioSAXS Guide | General Rad Safety |
| R-AXIS-IV Logbook | BioSAXS Logbook | nbs111 Rad Safety Rules |
| CCD Logbook | BioSAXS Manual | Radiation Hazards |
| Cryo how to | Proteum Manual | TX DSHS Radiation Safety | Crystal Cryo Log | Radiation Safety *Test* |
| ROBOTICS Policy | ||
| PHOENIX Quick Start | Zetasizer QS | Robotics *Test* |
| PHOENIX Manual | ZS Logbook | ZS Software Download |
| PHOENIX Logbook | Olympus-DP20 | Crystal Trak Trays |
| PHOENIX Sample Form | Minstrel Use | DIP User Manual |
| Sealing DWBs | Mass Spec. Cal. | DIP Logbook |
| HPLC Column Care | Nano Drop Log | |
| Administrative Docs | ||
| Emergency Plan | Faculty Use Form | Data archive info |
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Teachers: Please provide feed-back when using these materials in your courses. These courses are only published on the web. Please support this free publishing effort by registering your use. Educational users may download and edit these tutorials to suit their needs provided that the original author is given credit. Any comments or suggestions you may have to improve these tutorials will be greatly appreciated. Tutorials developed at UTMB:
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Table Of Contents
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Radiation Safety For X-ray Diffraction ∨ ∧ ⊗Safe Practices: Telltale Lights: Users should check the Power and Shutter lights every time they walk into the room. Each light operates in an interlock circuit such that if the light is off, you are guaranteed that either the Power is off, or the Shutter is closed. Beamstops: Beamstops serve two functions: to shorten the path of air scatter, and to protect the image plate. Users should check that the beamstop is in place whenever x-ray power is on, regardless of the shutter status. If you wish to remove the beamstop to check the direct beam position, you must remain in the room while you are doing the experiment. Protecting Your Fingers from the Direct Beam: Always CLOSE the shutters before working in the enclosures. When you manipulate anything in the vicinity of the goniometer, you should assume that the shutter is open and that there is no beamstop: therefore do not put your fingers in the path of the direct beam. Generator Room Access: Do not work behind the X-ray Generators. Generator rooms are not shielded from air scatter. Beam-confinement Labyrinths: Labyrinths are junctions between two pieces of equipment through which the beam passes. Any small disturbance of the x-ray optics can ruin the alignment, and reduce the beam intensity. In this event, call Mark White for assistance. Furthermore, if the equipment is severely distorted, dangerous amounts of x-rays can leak out. Visually check that the beam path is properly intact before opening the shutter. Survey Meters: Users should know how to conduct radiation survey with portable meter. X-ray Shutter: When data are not being collected the shutter switch should be closed. Before opening the shutter, double check that the beamstop and shields are in place, and that the labyrinths are properly aligned. Visitors: Visitors must be escorted by an authorized user. It is considered to be a risk factor to bring visitors into the generator rooms. |
RADIATION SAFETY FOR X-RAY UNITS ∨ ∧ ⊗NATURE OF ANALYTICAL X-RAYSAnalytical x-ray machines produce intense beams of ionizing radiation that are used for diffraction and fluorescence studies. The most intense part of a beam is that corresponding to the K emission of the target material and is called characteristic radiation. In addition to the characteristic radiation, a continuous radiation spectrum of low intensity is produced ranging from a very low energy to the maximum kV-peak setting. This is referred to as 'bremsstrahlung' or white radiation. Undesirable wavelengths may be filtered out using a monochromator. X-ray diffraction wavelengths (w) are selected so as to roughly correspond to the inter-atomic distances within the sample, and to minimize fluorescence. Wavelengths commonly used are 1.54 Å (Cu targets), 0.71 Å (Mo targets), 0.56 Å (Ag targets), and 2.3 Å (Cr targets). The relationship between wavelength and x-ray photon energy is determined by the equation
X-rays emitted from an open, uncollimated port form a cone of about 30 degrees. The x-ray flux can produce a radiation field at one meter on the order of 10,000 R/hr. A collimator reduces the beam size to about 1 millimeter diameter. X-RAY HAZARDS AND BIOLOGICAL EFFECTSX-rays produced by diffraction machines are readily absorbed in the first few millimeters of tissue, and therefore do not contribute any dose to the internal organs of the body. However, the lens of the eye can receive a dose from x-rays of this energy. Overexposure of lens tissue can lead to the development of lens opacities and cataracts. Absorbed doses of a few hundred rad may produce a reddening of the skin (erythema) which is transitory in nature. Higher doses -- 10,000 rad and greater -- may produce significant cellular damage resulting in pigment changes and chronic radiation dermatitis. Exposure to erythema doses may not result in immediate skin reddening. The latent period may be from several hours to several days. (Note: X-rays used for medical diagnosis are about one order of magnitude shorter in wavelength. Diagnostic rays are designed for tissue penetration and are carefully filtered to avoid x-ray damage to the skin caused by the longer, more readily absorbed wavelengths). SOURCES OF IONIZING RADIATIONThe primary beam is not the only source of ionizing radiation. Any high voltage discharge is a potential source of x-rays. Faulty high-voltage vacuum-tube rectifiers may emit x-rays of twice the voltage applied to the x-ray tube. Other sources of ionizing radiation are:
SAFETY PRECAUTIONS AND NOTESThe shielding, safety equipment and safety procedures prescribed for x-ray diffraction equipment are applicable only for up to 75 kV-peak x-rays. Additional or greater precautions are necessary for machines operating at higher voltages. The PI has the basic responsibility for providing a safe working environment by ensuring that equipment is operationally safe and that users understand safety and operating procedures. The equipment operator is responsible for his own safety and the safety of others when using an analytical x-ray machine. Prior to removing shielding or working in the sample area, the operator must check both the warning lights and the current (mA) meter on the console. Never trust a warning light unless it is on! Always use a survey meter to check that the shutters are actually closed if current is still being supplied to the tube. It is possible for a shutter to be stuck partially open even when the indicator shows that it is shut. The best way to avoid an accidental exposure is to turn the machine off before working in the sample area. Never put any part of the body in the primary beam. Exposure of any part of the body to the collimated beam for even a fraction of a second may result in damage to the exposed tissue. A person not knowledgeable about x-ray equipment should not attempt to make repairs or remedy malfunctions. If you suspect a machine is malfunctioning, turn it off or unplug it. Place a note on the control panel and inform the PI or his designated representative. Repairs to the high voltage section must not be made unless the primary leads are disconnected from the high voltage transformer and a signed and dated notice is posted near the x-ray ON switch. Turning off a circuit breaker is not sufficient. Bare feet are not permitted in the laboratory or around electrical equipment. Even slightly moist skin is an excellent electrical conductor and contact with faulty, ungrounded equipment may result in severe injury or death. Do not attempt to align x-ray cameras without first consulting an experienced person. Alignment procedures require special training and knowledge. Special care is required when one power supply is connected to more than one x-ray tube. EYE PROTECTIONThe use of safety glasses or prescription lenses is encouraged when working with analytical x-rays. While glasses cannot be depended upon to provide complete protection to the eyes, they can reduce x-ray exposure. Glass provides about 10 times the protection of plastic. Neither, however, will adequately protect the eye from direct exposure to the primary beam. FLUORESCENT SCREENSIt is unsafe to inspect an x-ray beam with a fluorescent screen without special precautionary measures. Notify the Safety Office before performing a procedure using a fluorescent screen. TUBE STATUS INDICATORSThere must be a visual indication located on or near the tube head to indicate when x-rays are being produced This is usually an assembly consisting of two red bulbs, wired in parallel and labeled X-RAYS ON. If one of the lights is burned out, the operator should either replace it before leaving the room, or leave a note on the light assembly indicating that the bulb is burned out. An unlit warning bulb does not necessarily mean that x-rays are not being produced. Always check the control panel. SAFETY DEVICESInterlock switches are used to prevent inadvertent access to the beam. They should not be bypassed. Interlocks should be checked periodically to insure that they are functioning properly. Interlocks and other safety devices and warning systems are not foolproof or fail-safe. A safety device should be used as a back-up to minimize the risk of radiation exposure -- never as a substitute for proper procedures and good judgment. |
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Detector Type |
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Detector Size image |
30 cm square |
9 cm square |
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Cycle time |
2 minutes |
3-20 sec. |
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Crystal-Det distance |
102 - 450 mm |
50 - 300 mm |
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X-Ray Source |
FR-E++DW 2.475 kW Cu/Cr 70 micron micro-focus |
MX06CE 1.2 kW Cu 100 micron ultra-fine focus |
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X-Ray Optics |
MSC Varimax Confocal Cu/Cr |
XENOCS Fox-2D |
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Cooling system -170 C |
100°K CryoCooler-II |
100°K CryoCooler-I |
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On-line training |
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Other Shared Instruments supported by the SCSB |
Malvern Zetasizer micro V Dynamic Light Scattering (Robotics Lab BSB 6.614) AUC (SBL MRB 5.148) BIACORE T100 (SBL MRB 5.148) |
MALDI Mass Spectrommeter (Solution Biophysics Lab MRB 5.148) THERMOFLUOR (SBL MRB 5.148) |
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Our R-AXIS IV++ is the primary X-ray area detector for macromolecular crystallography. Its two large active area imaging plates with fast readout speed and a wide dynamic range is combined with the FR-E++DW microfocus x-ray source, making it the most popular system for all aspects of macromolecular crystallography, from screening, to data collection, and phasing. |
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Data Collection See the R-AXIS-IV User Manual Instrument scheduling, for qualified users, is available online. |
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Our Brüker SMART-2K CCD X-ray area detector offers superior resolution for macromolecular and small molecule (drug) crystallography. Its fast 10s readout provides quick feed-back for initial screening. The 100micron fine-focus RAG x-ray source combined with the small 70 micron pixel size and 3D profiling, in SAINT, permits data collection on very long unit cells ~400 Å. |
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Data Collection See the SMART/SAINT/XPREP on-line how-to and the Proteum Manual Instrument scheduling, for qualified users, is available online. |
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Data Collection See the BioSAXS User Manual and the SAXNS group site. Instrument scheduling, for qualified users, is available online. |
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Instruction Manual See the X-ray Data Collection How-to.
Cryo-Loops See the Cryo-Loops How-to.
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| Our Cryo Industries of America (CIA) Cryo-Coolers offer one-button operation, fast cool-down times, low LN2 consumption, good temperature stability, and low sample temperatues. | ||||
Robotics Protocols
DLS & Nano-dropCrystallization Rooms & Incubator
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| MANAGER, CRYSTALLOGRAPHY | ∨ ⊗ | |
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Dr. Mark A. White
mawhite@utmb.edu Associate Professor of, Biochemistry & Molecular Biology, Manager, SCSB Macromolecular X-ray Laboratory Tel: 7.4747 |
Marked Map to X-ray Lab From Houston: Take the I-45 South to Galveston. Continue on the I-45 as it becomes Broadway. Turn Left on 14th street, at the Bishops Palace and St. Mary's Church. For the Mary Moody Thompson BSB continue until The Strand. Turn right onto The Stand, The BSB bulding (#54) is at the circle. Public Parking Garage #2 (directions) (#94) is on Harbor side drive, near 10th street. (UTMB Campus Map) |
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| CRYSTALLOGRAPHERS | ∨ ∧ ⊗ | |
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Dr. Kyung H. Choi
kychoi@utmb.edu Assistant Professor, Biochemistry & Molecular Biology |
Dr. Marc C. Morais
mcmorais@utmb.edu Assistant Professor, Biochemistry & Molecular Biology |
Dr. Stanley J. Watowich
watowich@xray.utmb.edu Associate Professor, Biochemistry & Molecular Biology |
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Dr. Y. Whtney Yin, MD
ywyin@utmb.edu Assistant Professor, Pharmacology & Toxicology |
Faculty Position Open
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| STRUCTURAL BIOLOGY ASSOCIATES | ∨ ∧ ⊗ | |
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Dr. Xiaodong Cheng
xcheng@utmb.edu Associate Professor, Pharmacology and Toxicology |
Dr. Edmund W. Czerwinski
ewczerwi@utmb.edu Emeritus Professor, Biochemistry & Molecular Biology |
Dr. Robert A. Davey
radavey@utmb.edu Assistant Professor, Microbiology |
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Randall M. Goldblum, M.D.
rmgoldbl@utmb.edu Professor, Biochemistry and Molecular Biology, Pediatric CEIID |
Dr. James C. Lee
jclee@utmb.edu Professor, Biochemistry & Molecular Biology |
Dr. Javier V. Navarro
jnavarro@utmb.edu Professor, Neuroscience & Cell Biology |
| PAST STRUCTURAL BIOLOGY ASSOCIATES | ∨ ∧ ⊗ | |
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Dr. Robert O. Fox
rofox@uh.edu Professor, Biology, UH |
Dr. R. Bryan Sutton
Roger.B.Sutton@ttuhsc.edu Associate Professor, Texas Tech HSC |
Dr. Irina Pikuleva
irina.pikuleva@case.edu Professor, Case Western Reserve |
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Dr. James R. Halpert
jhalpert@ucsd.edu Associate Dean, UCSD |
Dr. Emily E. Scott
eescott@ku.edu Associate Professor, University of Kansas |
Dr. Vincent J. Hilser
hilser@jhu.edu Professor, Johns Hopkins University |
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This group of Structural Biologists from several Gulf Coast Consortia instituions
has joined together to promote biological SAXS (BioSAXS) in the Greater Houston Area.
We have recognized the need to supplement our crystallographic, NMR, electron microscopy,
and biochemical data, with solution scattering. Our research interests cover a broad area,
but need additional information about our systems, such as: conformational rearangement, protein:protein or
protein:nucleic acid interactions, and the conformational ensemble of states, that can most
readily be determined via solution scattering.
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The Rigaku BioSAXS-1000 coupled with the FR-E++ x-ray source provides the best home-lab SAXS camera for biological samples. The SCSB's recent purchase of this system fulfills a regional need for BioSAXS instrumentation.
The SCSB research resources consist of several Center research labs designed to pursue intensive use of instrumentation by UTMB Structural Biologists, and thus are considered research facilities rather than fee-for-service cores. This includes the MXL BioSAXS facility. Facility users must supply their own materials and personnel associated with the measurement, otherwise there are no fees for users. In almost all cases, use of Center research resources is initiated either by a full member of the Center faculty or a Center Facility Lab Manager. Full Center faculty members (PIs) are entitled to engage in research of their own or in collaboration. However, before initiating a project ALL researchers must: (1) Discuss the project with a Center collaborator and (2) Sign a memorandum of understanding (MOU) for the project. The MOU outlines the role of Center and researcher. The MOU is usually created by the Manager for the collaborator.
To initiate a project in the Center please contact:
A Center
Faculty member
The
Facility Manager (Mark Andrew White, Ph.D.)
Or the Center Director, Monte Pettitt,Ph.D.
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What is PMB? PMB is a semi-automatic command-line interface to CNS. PMB also includes CNS modules and Patches which enhance it's capabilities and eases the structure-solution process. A few of the features of PMB are:
Why PMB? Well this started as a place to put programs being developed for the Gulf Coast Structural Genomics Consortium (GCSCC). Never heard of the GCSCC? Well that explains why PMB does not include any model-building tools. However, I started to include our automated structure refinement tools in the PMB suite, and the name PMB remains. The murky origin of this software lies deep within the ancient past of protein structure refinement with Alec Hodel and Paul Harkins of Robert Fox's group at Yale.
If you use these utilities please cite these papers:Andrew T. Russo, Mark A. White, and Stanley J. Watowich, The Crystal Structure of the Venezuelan Equine Encephalitis Alphavirus nsP2 Protease. Structure 2006 14: 1449-1458. (PDF)
Singh R, White MA, Ramana KV, Petrash JM, Watowich SJ, Bhatnagar A, Srivastava SK., Structure of a glutathione conjugate bound to the active site of aldose reductase. Proteins: Structure, Function, and Bioinformatics 2006, July 1, 64(1), 101-110; (PDF)
Emily E. Scott, Mark A. White, You Ai He, Eric F. Johnson, C. David Stout, and James R. Halpert, Structure of mammalian cytochrome P450 2B4 complexed with 4-(4 chlorophenyl)imidazole at 1.9 Å resolution: Insight into the range of P450 conformations and coordination of redox partner binding J. Biol. Chem 279, 26, 27294-301, 2004. (PDF)
A more detailed description of PMB and the improved CNS
routines included in pmb_bncs.f will be available when our Acta
Cryst. D paper is published.
Best Regards,
Mark A. White
mawhite@utmb.edu
If you use the fully hydrogenated or NQH-flipped features of J. Michael Word's
REDUCE program please cite:
Word JM, Lovell SC, LaBean TH, Taylor HC, Zalis ME, Presley BK, Richardson JS, Richardson DC.,
Visualizing and quantifying molecular goodness-of-fit: small-probe contact dots with explicit hydrogen atoms.,
J Mol Biol. 1999 Jan 29;285(4):1711-33 {PM}
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CNS 1.1 utilities. Last updated July 3, 2012. A typical installation will be placed in /usr/local/PMB (cd /usr/local ; tar -xzvf PMB_CNS_utils.tgz). Then add these lines to your .cshrc (/etc/csh.chrc) files: setenv PMB_HOME /usr/local/PMB/
setenv PMB_BIN $PMB_HOME/linux #OR# setenv PMB_BIN $PMB_HOME/irix #IRIX6.5 binaries are not maintained setenv PMB_BIN $PMB_HOME/mac_intel setenv PMB_BIN $PMB_HOME/mac_ppc Thanks to Evan R. Kantrowitz for providing the binaries for the MAC (OS 10.4).
The instructions on how to recompile CNS with the new restraint routines are located in the file: /.../PMB/CNS/other/pmb_bncs.f
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PMB_CNS13.tgz |
PMB-3: PMB with Patches for CNS1.3 (Coming Soon) |
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PMB_BRUKER_utils.tar.gz |
Bruker image and data processing |
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Macscience calibration utility |
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PMB_HKL_utils.tar.gz |
HKL utilities |
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Get old versions of PMB (not recommended) |
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An index of programs and utilities in PMB |
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| pmb_sca2cns pmb_CNS pmb_refine pmb_cns2phs pmb_start pmb_resolve pmb_check | |||||||||||||||
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The most current versions are installed on our main server in $PMB_HOME |
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This program converts a SCALEPACK merged HKL file into a CNS
format CV file with a 5% test set. The test set is picked using a
THIN SHELL algorithm, which is prefered when there is NCS. The
files Fobs.o.cv and Fobs.cv are also generated. This utility also
prints a $PMB_HOME/pmb_CNS command line for you to cut and
paste or edit, if the parameters need adjustment. Normally only
the high_res parameter may need to be changed, but check the
syntax of the "Space(Group)" entry. |
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pmb_CNS a b c alpha beta gamma "Space(Group)" SG_Number Resolution |
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This com file sets up ALL the CNS 1.1 input files using the given command line inputs. It also copies the necessary PMB refinement utilities into your current directory. The BINDIVIDUAL refinement script utilizes the improved B-factor restraints developed at UTMB by Mark A. White (Acta Cryst D submitted, 2006). Note the syntax of the "SpaceGroup" entry, which needs to be in quotes to be recognized as a single text string. {eg. P4(1), P2(1)2(1)2(1), C2(1), P3(1)21 } |
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pmb_refine -c5 -g -w2 -n -r -a -b -l -s22 -S -O -q -d -o -v -h [MODEL.pdb] |
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An automated CNS refinement program with several
command line flags:
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| -c{#} | (Cycles {opt#cycles}) -Default 1 cycle of refinement | ||||||||||||||
| -g | (Generate new topology files) - Do this after model building. Specify PDB file as last entry on line (1) | ||||||||||||||
| -w{#} | (Pick waters {opt#rounds}) - Default 1 round water pick | ||||||||||||||
| -n | (NCS-shake) - Relaxes NCS restraints for 1 round: "NCS Annealing" | ||||||||||||||
| -r | (Rigid body refinement) - Normally only used after MR | ||||||||||||||
| -a | (Simulated Annealing) - Do this with your initial models! (*See pmb_param.inp) | ||||||||||||||
| -b | (Generate & Use H-bond restraints) - For low resolution models (*See pmb_my_hbonds.inp) | ||||||||||||||
| -l | (LocalScale OFF flag) - Turns OFF local scaling: PHS files are still generated (2) | ||||||||||||||
| -s{#} | (Stereochem optimize ) - Optimize bond rmsd to Engh & Huber (*See pmb_param.inp) (3) | ||||||||||||||
| -S | (Stereochem Stretch & Relax ) - A Quick & Simple Annealing proceedure (3) | ||||||||||||||
| -O | (Stereochem Optimization Search ) - Find Optimal Bond RMSD Target (Optimizes X-ray Weight) (>Doubles refinement time!)(3) | ||||||||||||||
| -o | (Composit Omit Map) - Generate a Composit Omit Map after refinement | ||||||||||||||
| -q | (Occupancy refinement) - qindividual of selected atoms! | ||||||||||||||
| -d{%} | (Density Modification) - Use RESOLVE to generate a MAP (CCP4) (4)! | ||||||||||||||
| -v | (Validate structure) - Use PROCHECK & SFCHECK to check model(CCP4) (4)! | ||||||||||||||
| -h | (HELP) - View this help message | ||||||||||||||
Each refinement cycle continues until the freeR value stops
decreasing. This routine utilizes the Czerwinski/Matthews/Hynes
local scaling program to correct for absorption and anisotropic
scattering. It generates an XtalView format PHS file for generating a map in xfit or
COOT. The MTZ file new_sa.mtz generated with the -d flag (using pmb_resolve) is recommended for use in COOT.
(1) Run ./pmb_start on your rebuilt model OR specify your MODEL.pdb as the last entry on the command line. |
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A com file which launches the Czerwinski/Matthews/Hynes local scaling program pmb_cns2phs.I65, written in FORTRAN. This program uses the output of the CNS script FoFc_calc.inp (fo.sfo.fc.fob), which calculates Fc, Fbulk and phases, to produce an XtalView PHS format file (pmb_lscale.phs) and a corrected Fobs.cv (fobs_lscale.fob) file. The inclusion of the local scaling correction and bulk solvent component produces superior quality maps! |
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Configures your PDB file for generate to create the connectivity and stereo chemical restraints list. This splits your PDB file into the necessary components for generate.inp: start.protein.pdb, start.ion.pdb, start.ligand.pdb, start.nucl.pdb, start.carbo.pdb, and start.water.pdb. The start.ion.pdb and start.ligand.pdb files may need manual editing to insert the necessary TER cards between each ion or ligand. The new (2007) version of pmb_start is a more intelligent program. It automatically recognizes the standard amino acids, nucleotides, ions, carbohydrates, and waters. All other residues are now placed into the start.ligand.pdb file. A TER card is automatically placed between each ligand residue as the residue number or chain_id changes. This requires that the atoms of a residue are contiguous within the PDB file. This function is now called if you specify the PDB file at the end of the "pmb_refine -g ... My_Model.pdb" command. |
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Create a solvent-flipped map (CCP4+RESOLVE). This runs RESOLVE using your latest model (minimize.pdb) and phases (pmb_lscale.phs). The MTZ files new_sa.mtz and resolve.mtz generated by pmb_resolve are recommended for use in COOT. This script is run after refinement with the pmb_refine -d{%sol} flag. Users may enter an optional %solvent value, or let CNS estimate the solvent content. BUG: Requires CCP4. |
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Check the Quality of your Model (CCP4) This runs both PROCHECK and SFCHECK on your latest model (minimize.pdb). This script is run after refinement with the pmb_refine -v flag. BUG: Requires CCP4. Note: The Molprobity reduce program is always run at the completion of any refinement. It is a good idea to look at the YYMMDDx.##.Project-flipped.pdb for possible His/Asn/Gln flips. |
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generate.mtz(pdb), minimize.pdb, bindividual.pdb |
The output files of generate.inp, minimize.inp and bindividual.inp. They are used as the input files for iterative minimization/b-factor refinement. The pmb_param.inp file can override the defaults set in these input files: (minimize, bindividual, anneal) |
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Fobs.o.cv, Fobs.cv |
The ORIGINAL CV format data file, and the locally scaled file used by most refinement scripts. |
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start.protein.pdb, start.ion.pdb, start.ligand.pdb, start.water.pdb, start.nucl.pdb, start.carbo.pdb |
The input files for generate containing your protein model, the ions, any ligand, and the waters. |
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ligand.top, ligand.param |
These ligand parameter and topology files must exist in your working directory. They may be empty if you do not have a ligand. (Look in $PMB_HOME/CNS/other for some examples) |
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A roughly cronological order of how to use the PMB utilities |
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Convert Scalepack {SCA} intensities to CNS Fobs {CV} with 5% test set |
Run $PMB_BIN/pmb_sca2cns ScalePack.sca to generate your Fobs.cv file. This utility also copies the output file "ScalePack.sca.cv" to the two required CV files for you: Fobs.o.cv and Fobs.cv |
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Make the CNS input files |
Run $PMB_HOME/pmb_CNS a b c alpha beta gamma “S(G)” SG_No. Res to automatically produce the required CNS input files and copy all the PMB utilities into your current directory. Use this command to override the spacegroup in the SCA file or to change the resolution limit. Warning: This clobbers all of your CNS INP files and the ligand toppolgy/parameter files. |
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Set up PDB for CNS |
Then run "pmb_start My_Model.pdb" to set up your CNS input models. The start.ion.pdb file may need manual editing to insert the necessary TER cards between each ion. The script pmb_start has recently been changed to a more intelligent perl script. Unidentified residues are now added to the start.ligand.pdb file with TER cards placed at the end of each residue. |
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Refine your model in CNS |
To refine the model and generate a map run: |
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Build the Model |
XtalView may be run using the "sharefonts;xtalmgr &" command.
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Use NCS and Solvent Flattening in RESOLVE/CCP4 |
Edit pmb_resolve to have the correct CELL and SG number. Pick suitable residues to use as the basis of NCS and edit the default selection criteria accordingly. Note that you do not want too many nor too few atoms to be selected. Note that this script requires that the model has been refined at least once in order to generate PHIC. |
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Convert Scalepack to CNS CV |
$PMB_BIN/pmb_sca2cns My_Data.sca |
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Set up files for CNS |
$PMB_HOME/pmb_CNS 47.812 47.812 62.134 90.000 90.000 90.000 "P4(1)" 76 1.7 |
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Prepare PDB model for CNS |
pmb_start 1STN.pdb |
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Refine your model in CNS |
pmb_refine -g -r -a -s 1STN.pdb |tee MMDDHH_My_Project_log |
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Build the Model in XTALVIEW |
sharefonts;xtalmgr &
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Use NCS and Solvent Flattening in RESOLVE |
pmb_resolve |
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FAQs ^ |
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Automated Refinement «^v»PMB helps to automate the refinement process through the pmb_refine command line script. This program accepts many flags to customize the refinement process. All refinements are run to convergenge as determined by the R
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Improved Stereochemistry ^PMB includes modified TOPOLOGY and parameter file for proteins. These changes were based on our experience with high-resolution strucuture refinement using the Eng & Huber bond rmsd targets (see below). After every refinement is finished the REDUCE program (part of MOLPROBITY) will create a fully hydrogenated model and check for possible NQH rotomer flips. These models are saved in the MMDDHH.XX.ProjID+H.pdb & MMDDHH.XX.ProjID-flipped.pdb files. | |||||||||||||||
Optimized Weights ^ | |||||||||||||||
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PMB will automatically calculate the optimal weights for refinement. This will benefit refinements of either high- or low-resolution structures. The optimal weight will neither over- or under- weight the xray terms.
Pictured on the left is an example of the automatic optimization data produced with the -O flag. Just look for the PNG image in your directory to see the entire optimization plot. The software will select the optimum bond-target based on this data and use it for refinement. |
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MAP Generation ^PMB helps to automate the map generation process.
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B-factor Patches ^ | |||||||||||||||
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PMB contains a CNS module, pmb_bncs.f, which removes much of the model-bias from B-factor refinements. This isotropic B-factor model seems to work at any resolution. This is because it inherently modifies the restraints to match the apparent resolution of the data. Users are still provided with a bdomain.inp CNS input file, however the bindividual.inp script is prefered when using the PMB/CNS B-factor patch. | |||||||||||||||
| B-factor plot of 1AV1, refined at 4 Å using the PMB B-factor patch for isotropic B-factor restraints. Note that even the side-chain b-factors are well-behaved, and that the molecular motions are modeled more accurately by these individual B-factors than they could be by a series of group B-factors. | ||||||||||||||
Improved NCS ^PMB has several features for the better use of NCS. An improved NCS B-factor patch allows each NCS group to "float" with a group B-factor, better modeling variation in the local enviroment of each NCS domain. The NCS annealing feature has been demonstrated to lower the Rfree by allowing the structure to move out of a local minimum trap more readily. PMB will also generate NCS averaged maps for you (see above). Also, See the pmb_refine -n flag entry. | |||||||||||||||
Low Resolution Refinement ^PMB greatly improves refinement at low resolution. Some of the improvements offered for low-resolution structures include: | |||||||||||||||
CONTACT:Mark A. White,© 2001-2013 |
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PUBLICATIONS | Last Update: Jan, 2013. |
| These are Structural publications from the Sealy Center for Structural Biology and Molecular Biophysics Macromolecular X-ray Laboratory faculty and staff. | |
2013 ∨ ∧ ⊗ |
PubMed 2013 |
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Haijun Chen, Chunyong Ding, Christopher Wild, Huiling Liu, Tianzhi Wang, Mark A. White, Xiaodong Cheng, Jia Zhou, Efficient Synthesis of ESI-09, A Novel Non-cyclic Nucleotide EPAC Antagonist, Tetrahedron Lett. 2013 54 1546-1549. Epub: Jan. 21 {PM} {PDF} |
2012 ∨ ∧ ⊗ |
PubMed 2012 |
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White MA, Li S, Tsalkova T, Mei FC, Liu T, Woods VL Jr, Cheng X., Structural analyses of a constitutively active mutant of exchange protein directly activated by cAMP., PLoS One. 2012;7(11):e49932. Epub: 2012 Nov 26. {PM} {PDF} |
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Bussetta C, Choi KH., Dengue virus nonstructural protein 5 adopts multiple conformations in solution. Biochemistry. 2012 Jul 31;51 (30):5921-31 Epub: Jul 16 {PM} {PDF} |
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Meiling Lu, Yan Huang, Mark A. White, Xuri Wu, Nan Liu, Xiaodong Cheng and Yijun Chen, Dual catalysis mode for the dicarbonyl reduction catalyzed by diketoreductase Chem. Commun. 2012;48: 11352-11354 {PM} {PDF} |
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Zheng J, Gay DC, Demeler B, White MA, Keatinge-Clay AT., Divergence of multimodular polyketide synthases revealed by a didomain structure. Nat Chem Biol 2012 964 Epub: May 27 {PM} {PDF} |
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Morais MC., The dsDNA packaging motor in bacteriophage φ29., Adv Exp Med Biol. 2012;726:511-47. Review. {PM} {PDF} |
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Choi KH., Viral polymerases., Adv Exp Med Biol. 2012;726:267-304. Review. {PM} {PDF} |
2011 ∨ ∧ ⊗ | PubMed 2011 |
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Sheng Li, Tamara Tsalkova, Mark A. White, Fang C. Mei, Tong Liu, Daphne Wang, Virgil L. Woods Jr. and Xiaodong Cheng Mechanism of Intracellular cAMP Sensor Epac2 Activation: cAMP-induced conformational changes identified by peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS). J Biol Chem. 2011 286(20);17889-97 Epub: March 17 {PM} {Review} {PDF} |
2010 ∨ ∧ ⊗ | PubMed 2010 |
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Russo AT, Malmstrom RD, White MA, Watowich SJ. Structural basis for substrate specificity of alphavirus nsP2 proteases. J Mol Graph Model. 2010; 29(1);46-53 Epub]: Apr 24 {PM} {PDF} |
2009 ∨ ∧ ⊗ | PubMed 2009 |
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Tsalkova T, Blumenthal DK, Mei FC, White MA, Cheng X. Mechanism of EPAC activation: Structural and functional analyses of EPAC2 hinge mutants with constitutive and reduced activities. J Biol Chem. 2009; 284(35);23644-51 Epub: Jun 24 {PM} {PDF} |
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Fuson KL, Ma L, Sutton RB, Oberhauser AF, The c2 domains of human synaptotagmin 1 have distinct mechanical properties, Biophys J. 2009; 963);1083-90 EPUB: {PM} {PDF} |
2008 ∨ ∧ ⊗ | PubMed 2008 |
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Mark Andrew White, Natalia Mast, Ingemar Bjorkhem, Eric F. Johnson, C. David Stout, and Irina A. Pikuleva, The Use of Complementary Cation and Anion heavy-atom salt derivatives to solve the structure of cytochrome P450 46A1, Acta Cryst. D 2008; 65(16);487-95 EPUB: April 20. {PM} {PDF} |
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Natalia Mast, Mark Andrew White, Ingemar Bjorkhem, Eric F. Johnson, C. David Stout, and Irina A. Pikuleva, Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principle cholesterol hydroxylase in the brain, PNAS 2008; 105(28);9546-9551 EPUB: July 9. {PM} {PDF} |
2007 ∨ ∧ ⊗ | PubMed 2007 |
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Fuson, K.L., Montes, M., Robert, J. J., Sutton R. B., Structure of Human Synaptotagmin 1 C2A-C2B in the Absence of Ca2+ Reveals a Novel Domain Association, Biochemistry 2008; 46(45);13041-13048 EPUB: Oct. 23. {PM} {PDF} |
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Zhao Y, Sun L, Muralidhara BK, Kumar S, White MA, Stout CD, Halpert JR., Structural and Thermodynamic Consequences of 1-(4-Chlorophenyl)imidazole Binding to Cytochrome P450 2B4. Biochemistry 2007 Oct 16;46(41):11559-67. EPUB: Sept 22. {PM} {PDF} |
2006 ∨ ∧ ⊗ | PubMed 2006 |
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Aditya Hindupur, Deqian Liu, Yonghong Zhao, Henry D. Bellamy, Mark A. White, and Robert O. Fox, The crystal structure of the E. coli stress protein YciF. Protein Science 2006 EPUB: Sept 25. {PM} {PDF} |
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Andrew T. Russo, Mark A. White, and Stanley J. Watowich, The Crystal Structure of the Venezuelan Equine Encephalitis Alphavirus nsP2 Protease. Structure 2006 14: 1449-1458. {PM} {PDF} |
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Montes M, Fuson KL, Sutton RB, Robert JJ. Purification, crystallization and X-ray diffraction analysis of human synaptotagmin 1 C2A-C2B. Acta Cryst. F 62, Page 926-9, Aug 2006. {PM} {PDF} |
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Singh R, White MA, Ramana KV, Petrash JM, Watowich SJ, Bhatnagar A, Srivastava SK., Structure of a glutathione conjugate bound to the active site of aldose reductase. Proteins: Structure, Function, and Bioinformatics 2006, July 1, 64(1), 101-110; [April 25, Epub ahead of print] {PM} {PDF} |
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Mukherjee M, Dutta K, White MA, Cowburn D, Fox RO., NMR solution structure and backbone dynamics of domain III of the E protein of tick-borne Langat flavivirus suggests a potential site for molecular recognition. Protein Science 2006, June 15(6), 1342-55. {PM} {PDF} |
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Russo AT, Watowich SJ, Purification, crystallization and X-ray diffraction analysis of the C-terminal protease domain of Venezuelan equine encephalitis virus nsP2. Acta Cryst. F 62(6), Page 514-7, June 2006. {PM} {PDF} |
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Zhao Y, White MA, Muralidhara BK, Sun L, Halpert JR, Stout CD., Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: Insight into P450 conformational plasticity and membrane interaction. J Biol Chem. 2006, March 3, 281(9), 5973-81; [Epub ahead of print: Dec 21] {PM} {PDF} |
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Sutton RB, Vishnivetskiy SA, Robert J, Hanson SM, Raman D, Knox BE, Kono M, Navarro J, Gurevich VV., Crystal structure of cone arrestin at 2.3A: evolution of receptor specificity., J Mol Biol. 2005 Dec 16;354(5):1069-80. Epub 2005 Nov 2. {PM} {PDF} |
2005 ∨ ∧ ⊗ | PubMed 2005 |
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Schultz DA, Friedman AM, White MA, Fox RO., The crystal structure of the cis-proline to glycine variant (P114G) of ribonuclease A, Protein Sci. 2005 Nov; 14(11):2862-70. [Epub ahead of print; Sep 30] {PM} {PDF} |
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Bi-Hung Peng, Mark A. White, Gerald A. Campbell, Jebamony J. Robert, J. Ching Lee, Roger B. Sutton, Crystallization and preliminary X-ray diffraction of the ZO-binding domain of human occludin Acta Cryst. F 61, Page 369-371, Apr 2005. {PM} {PDF} |
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Czerwinski EW, Midoro-Horiuti T, White MA, Brooks EG, Goldblum RM., Crystal structure of Jun a 1, the major cedar pollen allergen from Juniperus ashei, reveals a parallel beta-helical core. J. Biol. Chem 280, 5, 3740-6, 2005. {PM} {PDF} |
2004 ∨ ∧ ⊗ | PubMed 2004 |
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Liu D, Zhao Y, Fan X, Sun Y, Fox RO., Escherichia coli stress protein YciF: expression, crystallization and preliminary crystallographic analysis. Acta Cryst. D 60, Page 2389-90, Dec. 2004. {PM} {PDF} |
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Emily E. Scott, Mark A. White, You Ai He, Eric F. Johnson, C. David Stout, and James R. Halpert, Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9 A resolution: Insight into the range of P450 conformations and coordination of redox partner binding J. Biol. Chem 279, 26, 27294-301, 2004. {PM} {PDF} |
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Liu D, Zhao Y, Fan X, Sun Y, Fox RO, Expression, crystallization and preliminary crystallographic analysis of YciE, a stress protein from Escherichia coli. Acta Cryst. D 60, Page 1888-9, Oct. 2004. {PM} {PDF} |
2003 ∨ ∧ ⊗ | PubMed 2003 |
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Yonghong Zhao, Deqian Liu, Warna D. Kaluarachi, Henry D. Beleamy, Mark A. White, and Robert O. Fox, (2003), The crystal structure of Escherichia coli heat shock protein YedU reveals three potential catalytic active sites, Protein Science 12, 2303-11, 2003. {PM} {PDF} |
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Emily E. Scott, You Ai He, Michael R. Webster, Mark A. White, Christopher C. Chin, James R. Halpert, Erik F. Johnson, and C. David Stout, (2003), An open conformation of mammalian cytochrome P450 2B4 at 1.6-A resolution, PNAS 100, 13196-13201, 2003. {PM} {PDF} |
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Mark A. White, Deqian Liu, Michael R. Holbrook, Robert E. Shope, Alan D. T. Barrett and Robert O. Fox, (2003), Crystallization and preliminary X-ray diffraction analysis of Langat virus envelope protein domain III, Acta Cryst. D 59, 1049-51, 2003. {IUCr} {PDF} |
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Deqian Liu, Terumi Midoro-Horiuti, Mark A. White, Edward G. Brooks, Randall M. Goldblum and Edmund W. Czerwinski (2003), Crystallization and preliminary X-ray diffraction analysis of Jun a 1, the major allergen isolated from pollen of the mountain cedar Juniperus ashei, Acta Cryst. D 59, 1052-53, 2003. {IUCr} {PDF} |
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Landau EM, Pebay-Peyroula E, Neutze R. Structural and mechanistic insight from high resolution structures of archaeal rhodopsins. FEBS Lett. 555(1), Page 51-6. Nov 27, 2003. {PM} {PDF} |
2002 ∨ ∧ ⊗ | PubMed 2002 |
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Crystallization of membrane proteins in cubo., Nollert P, Navarro J, Landau EM., Methods Enzymol. 2002;343:183-99. {PM} {PDF} |
2001 ∨ ∧ ⊗ | PubMed 2001 |
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John O. Wooll, Robert H. E. Friesen, Mark A. White, Stanley J. Watowich, Robert O. Fox, J. Ching Lee* and Edmund W. Czerwinski*, (2001), Structural and Functional Linkages Between SubunitInterfaces in Mammalian Pyruvate Kinase, J. Mol. Biol. 312, 525-540, 2001. {PM} {PDF} |
2000 ∨ ∧ ⊗ | PubMed 2000 |
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Kinetic and structural characterization of the glutathione-binding site of aldose reductase., Dixit BL, Balendiran GK, Watowich SJ, Srivastava S, Ramana KV, Petrash JM, Bhatnagar A, Srivastava SK., J Biol Chem. 2000 Jul 14;275(28):21587-95. {PM} {PDF} |
1999 ∧ ⊗ | PubMed 1999 |
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Mark A. White, Stanley J. Watowich, and Robert O. Fox, (1999), Calibration of Spiral Readout Image Plate Detectors, J. Appl. Cryst. 32, 65-70, 1999. {IUCr} {PDF} |
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