G. Wagner Comment - Can detect dimer unambiguously by 15N-H1D <-> 14N-H H mixtures
ONLY see for dimer
June 6, 1999
Jeffrey Hoch "Opening Remarks"
David Case "Where do we go from here? Some hard problems in determining biomolecular structures and dynamics"
Lewis Kay "Experimental NMR Spectroscopy: A Brief Survey of an Evolving Field"
June 7, 1999
"Modern Spectrum Analysis in Multidimensional NMR: A Critical Reassessment"
maxEN w/ expon. sampling in two dimensions provided better S/N than LP/DFT or DFT alone. S/N Issue
Errors: false peaks, undetected peaks *see Rowland NMR Toolkit - for generating simulated NMR spectra
Nice summary of advantages and disadvantages of LP and MaxEN
Timothy Havel "A matrix least-squares approach to estimating distance constraints from NOESY spectra"
Good project for David Snyder: Havel et al. (1994) PNAS 91: 7962 (could not read easily) 2D spectra can be factorized into 3 spectra
getting many J(H-H) // Yang & Havel (1994) J. Biomol. NMR 4: 807; Yang et al. J. Biomol. NMR 4: 827
Also compared at end with Autopsy
NOESY- peak matrix generated from COSY (i.e. could be done from chemical shift peak list) and then extract out the peak amplitudes.
The formulation yields all intensities and does not require you to assign peaks themselves
Accurate peak shape model
Accurate chemical shifts
Need also correct linewidths and multiplicities (which he gets from COSY)
Using "noise flattening" algorithm from AUTOPSY program - and "eigen filtered"
I think this could work much better if you (1) use 13C-edited
NOESY (2) pick high - determine structure-use structure to pick lower
He is getting very nice peak picked NOESY spectra
"Combined automated assignment and 3D structure calculations of proteins:
Does it work in practice?"
CsE - vs
from Ram Krish - homology NOESY/TOCSY
used coupling constants as constraints
No manual assignments used as input
Made comparison between manual and automatically determined constraint lists
Major issue is peak picking - some significant differences between manual / automated structure analysis
Using homologous structure to drive automated NOESY analysis
Get correct fold - but not outstanding results helix tilt is pretty far off
Gaetano Montelione "Automated Analysis of Protein NMR Spectra: Prognosis of Structural Genomics"
Frank Delaglio "Things a Nice Italian Boy Can Do With Couplings, Shifts, and Molecular Dynamics"
13C db for x-ray structures of resolution < 2.2 Å
Obtained from cross-correlation - 2910 predicted
Shifts 1920 correct
problem -> 58 incorrect
Bruce Johnson "NMRView: New tools for assignments and NOE analysis"
"NMR without NOEs: Getting the most from your Chemical Shifts"
Important that we fish out all assignments and get to bmrdb
- NMR meets Bioinformatics
- can use Chemical Shifts like sequence data
380 sets of assignments in BMRDB
1100 distinct structures
Assignment of Protein A ---------> Assignment of Protein B
ORB Gronwald et al. (1997) JBNMR 10: 165 Transferring
GARAMM JBNMR 7: 207 homologous
SHIFTY Wishart et al. (1997) JBNMR 10: 329 assignments
SHIFTY - like what we were trained to do with Norma ~1990 (access from BMRDB)
correlation coefficient of > 90% when 90 sequence identity > 35%
- using HNCO/HN(CA)CO get ~90 - 100% ASSIGNMENTS
< 100 residues TOCSY
75 - 150 residues TOCSY - HSQC | experiments
75 - 200 residues CBCA(CO)NH | to provide
> 200 residues HNCO / HN(CA)CO | assignments
should be |
SHIFTX - computer 1H chemical shifts from
running this | 15N referencing big problem in db
routinely on | but can get r's of ~ 0.92 - good way to validate
all assignments/| data referencing 13Ca, 13C'abo good a
3D structures | Ca shift related to disulfide conformation
Discuss with Hunter:
He is using very simple spectra + d predicted from crystal structures to complete assignments for case where crystal structure is known.
Also- using homology model to predict d and then use these together with minimal experimental data to complete assignments.
Trying to use SIMPRED to predict assignments and then combine with simple TOCSY or TR experiments.
1) Measure d
2) Initial secondary structure from d's
3) Fold using GA + threading
4) Energy minimization
Wants to apply to make assignments in bound vs. free ligand binding
* need to get someone working on this idea that we have already discussed alot
Angela Gronenborn "Structures of Protein-Protein and Protein-DNA Complexes"
could use conformational db in initial fold calculation - then eliminate in subsequent refinement
- record as much data as we can lay our hands on
- 5 months of measure time for 40 hD complex - complex too weak to crystallize
June 8, 1999
Rafael Brüschweiler "Anisotropic Motions Studied by NMR relaxation, MD, and DFT"
It is impossible to distinguish overall motion from internal motions - this is a consequence of the inability to distinguish multiple motions with different amplitude that occur on the same timescale - Jardetsky
approach | computer simulation | motional
spectral density | Normal model | models
mapping | analysis |
MD - simulations guided model building - best we can do - Recent work from Ernst lab
Long MD simulation statistical analysis
Explained problems is setting CSA tensors from ssNMR, and liquid state
NMR - proposed using quantum mechanical calculations (DFT - Density Functional
Theory) to get these CSA tensors.
-very good agreement between calculation and ssNMR values in amino acid models
CSA tensions are changing (rapidly) during the MD trajectory - we usually consider these to be static but they are actually fluctuating
3J(15N-13C') coupling (~1 - 2 Hz) have been measured in ubiquitin (S. Grzesiek published recently in JACS)
Gerhard Wagner "NMR studies of protein interactions"
Nice example of using mutation to enhance properties of proteins - stability of aggregation
CD2: carbohydrate essential for stabilizing protein structure (maybe true
in other cases) - but can make mutant form which is stable without
sugar - this is published already Wyss, Endo
Should look for sugar binding sites in parl - look for consensus carbohydrate binding sites
CD58: homology model should surface exposed loops with hydrophobic groups - mutated 6 of these to hydrophilic - now get great spectra - just looked for surface hydrophobics.
Ad Bax "NMR of weakly aligned macromolecules"
using 3JHC to identify H-bond pairs!!!! Discuss with Dani
Very nice experiment: DNH, DNC, DC'H all measured simultaneously in 30 kD protein - provides orientation of peptide planes
Q=[(S(Dcalc - Dobs)2)/S(Dobs)2]1/2
can be used as a cross validation
For bb- see good agreement between calculation (from x-ray) and observed D's. But for side chains, not a good agreement - this has been related to dynamics. Motional average causes dipolar coupling to be smaller than true value - used in XPLOR as "lower bound"
Aligning a protein using purple membrane fragments to measure dipolar couplings - bacteriorhodopsin (BR)
Bicelle orientation tensors different from BR orientation tensor - get intersections of cones
use 591 NOEs, 945 Dipolar couplings, 90 torsional constraints provides good quality structure - but make difficult to get convergence
Measured N-H bond length from NMR data
GNH =1.041 ± 0.006 Å
"An order matrix approach to the structural and dynamic analysis of residual
dipolar couplings in biomolecules"
to brielle mixture
"Chromatic NMR relaxation approach to structure and dynamics of proteins"
Field dependent relaxation measurements
Multiple field (3 fields) => parameter of spectral density functions
360, 520, 500 data for ubiquitin
CSA - 120
- 200 ppm surprisingly wide
q = 6 - 26 ° similar measurements have been made for otherproteins
See Fuschman et al (1998) JACS 120: 10947 for a discussion of how CSA uncertainties propagate to dynamic parameters. Also (1999) J. B. NMR 13: 139
Sandor Szalma "Methodologies for NMR Structure Refinement: Direct Refinement Using NOE Intensities, Residual Dipolar Couplings, Cross-correlated Relaxation Rates and Local Ensemble Averaging"
see Haenggi & Braun (1994) FEBS Letts. 344:147 Removal of violations
Have implemented cross-correlation relaxation constraints (G) and residual dipolar coupling measurements in XPLOR. Also, implemented ensemble averages of 3J, G and NOEs.
Michael Nilges "Optimal use of NOE restraints"
Ambiguities handled differently from other applications
*Send Nilges CONGEN papers (*loose bounds *shape of potential function) he is using Donna's potential, should point this out
This idea previously done by Constantine et al.: Use violation analysis to identify inconsistent constraints - identify violated constraints - ensemble average in this region of molecular (e.g. Constantine et al.) - but ensemble averaging is done with cross validation.
Run MD unconstrained
compute <dij>, compute sij
from autocorrelation of tdisx
vs dis from true NOE data
calibration with min. at distances calibrated with NOE
deposit NOESY peaklists, not upper bounds
used MD simulations to help in interpreting of NOE data???? did not understand
"Torsion angle dynamics in automated NMR structure calculations"
DYANA - torsion angle dynamics
CAR / DYANA - introduce ambiguous constraints, introduce simulated annealing
For each peak - discard assignments contributing £ Pvol % to peak volume
Recalibrate distances assuming multiple contributes to each peak
Each cycle starts from original unassigned peak list
Robust with artifactual peaks
apo CopZ Cu-binding protein Wimmer et al. in press
manual vs. automated analysis | only running 6 cycles
* shows bundles at each of 6 stages - happy that fold is defined already at 1st cycle
* shows cf to manual structure at each stage
only ~1% of difference is
assignments between manual
- Imperfect peak picking-artifactual peaks-impurities
Methods to improve reliability:W. Braun - need to define reliability index for quality of assignments.
* use of manual assignments
* criteria for reliable peaks
* multiple assignments
Using "contact" frequency to define peak assignment reliability. Mutually supporting NOE assignments
Ambiguous Distance Restraints: The presence of wrong assignment possibilities have little influence on the structures so long as the correct one is on the structure
Idea: From two peaks end (e.g. selected randomly) combine assignments into one extended multiple restraints
eg.1. N peaks - 10% wrong, only 1% contribution???
eg. 2 P14a compare of CAR, X-ray, manual about as good/bad as our results
J's input, constraints but dihedral constraints computed automatically Used manual assignment peak list - clear peak list. But another protein - Killer toxin- using Autopsy peak list.
June 9, 1999
Wilfred van Gunsteren
"The effect of motional averaging on the calculation of NMR-derived structural
* MD simulation of b heptapeptide - ensemble average satisfies NOE and J constraints better than any single NMR structure (see Sept issue of Proteins)David Pearlman "Improved Methods for NMR Structure Determination from NOE-derived Distances and Coupling Constants"
using 50 ns MD simulation at 340° K
J. Mol. Biol. (1998) 280: 925 - 932
75 % unfolded -> no NOE violation larger than 1 Å on average
J values not good indications of folded / unfolded equilibrium
NMR data has only limited capacity to distinguish - single fold from mixture A folded/unfolded conformers
Peter Wright - also feels that J's are generally not good indicators of folded/unfolded - except when you see extreme J's (e.g. ~3 Hz)
Nice idea (shows advantages over Torda - type time average): FINGAR - Fit NMR using Genetic AlgorithmMichael Levitt "Accurate Molecular Dynamics Simulation for NMR"- define conform database (use MD and / or PG + cluster)Extended FINGAR: finds bad restraints. FINGARW does a good job of tagging bad restraints - but I did not understand this???
- to each conf assign weight
- use genetic algorithm to determine weights
Also showed example of discovering bad restraints
Argues that FINGAR is difficult for proteins because hard to generate basis set. Maybe can generate rotomer states in regions of protein with residual violations. - Could compute combinatorial number of rotomer states.
1 ns / day for MD ?Lucia Banci "Computational Aspects in NMR Spectroscopy of Paramagnetic Metalloproteins"
NO NOTESKathleen Hall "Unrestrained stochastic dynamics simulations of the RNA UUCG tetraloop using an implicit solvation model"
NO NOTESJoseph Puglisi "Solving biologically relevant RNA structures by NMR"
NO NOTESJames Williamson "Approaches to NMR of Large RNAs"
NO NOTESJuli Feigon "Cation Binding, folding and interactions in DNA and RNA"
NO NOTESJune 10, 1999
"Structural and dynamical characterization of disordered states of proteins"
Unfolded proteins have functional rules
Kinase-induced activation domain (pKID) of CREB.
- 1H - 15N HSQC looks like unfolded protein - but see significant spectral changes upon binding to target protein
- Should review these papers: pKID : KIX complex
60 AA peptide fragments at 180 residue proteins
80 residue fragments fully functional
not folded - because of truncation of C- terminal helix
active and folded
maybe dmPI is cut wrong
-make it longerPoplar pastocyanine
- folded at high salt (1M NaCl)* limited resonance dispersion
- unfolded at low salt
* tendency to aggregate (need to work at 100 - 300 mM)
* variable linewidths
* ensemble averaged NMR parameters
NMR Parameters in Unstructured Proteins
important - chemical shifts | coupling constants not very useful
Discussion with Hunter - use CO-TOCSY, CA-TOCSY, etc to do complete assignments
Christian Griesinger "Computational Aspects of novel NMR parameters for the determination of structure and dynamics of biomolecules"
Peptide - C20W - calamodulin complex
Yb (not the best - distortion? would be better. Dp?; will give both magnetic susceptibility and pseudoconstant shifts) - DTPA (buy an anhydric and couple to N-term of peptide) - Peptide - (this peptide is then bound to calamodulin)
gets residual dipolar coupling and pseudoconstant shifts
gets relative orientation of two domains (using order matrix approach for using dipolar coupling data)
Use of dipolar coupling as intervector projection constraints - take relative orientation of N-H bind vectors - form allowed / disallowed 0 values.
using easily accessible structural info for structural bioinformatics
Can thus use restraints as local restraints right from start - do not need ? orientation
combine 13C C5I and dipolar couplings - use as constraints to try to map protein to structures in the PDB (!!! locations and relative orientations of helices)
Idea: Can the dipolar coupling in Z-domain sort out of helix-1 distortion - can also consider to use chemical shift changes in orientation as structural constraints
Christina Redfield "Structure and Dynamics Probed by 15N Relaxation and Partial Sample Orientation"
Old data - the average errors for T1, T2 were ~1.5%
assume isotropic tumbling
2)Need to account for a ~20°
take into account effort of non-colinearity:
at 500 MHz - less important efforts are more pronounced at higher
at 750 MHz ~2% magnetic fields and with more
at 900 MHz ~4% anisotyping
bundles: 5% DMPC:DHPC
2.9:1.0 also with
7.5% DMPC:DHPC 2.9:1.0 charge - minimize
7.8% DMPC:DHPC:CMB 2.9:1.0:0.1
with CTAB get orientation ? that matches. inertia moments, without CTAB set tilt due to electrostatic effects. Having two different allignments useful to fit CSA ?.
for axially ? CSA ? - also did analysis for asymmetric CSA tensor - similar
using s=1, rNA=1.04, DJ=-174.4, hasy=0.14, a=18.6°
s=0.94, rNH=1.02, DG=-172.4, h=0.14, a=18.7°
Hartmut Oschkinat "Steps towards automating the structure determination process and concepts for membrane protein structures"
Deborah Kallick "Analysis of Proton Chemical Shifts in Turns"
John Markley "News from NMRFAM and BMRB: Experiment Management System for Biomolecular NMR and Software Tools for Archiving and Retrieving NMR Depositions"
* Role of NMR in Structural / Functional Proteomics
Management System for NMR
Lab organization - expression vectors
Data processing / analysis
Comprehensive definity tags for NMR_Star is in submission forms for BioMagResDatabase
May be as
many as 400 new structures in 1999.
Projections: 2000 503 structures
2001 686 structures
* Make use of XML as alternative format to NMT-Star
* Develop CORBA interfaces to the databases
SeSene - NMRFam experiment management system. Experiment management system for NMR
student look at this)- available from BioMagResDatabase Server (under software)
Camel Client / Alibaba Server
can archive experimental protocols (e.g. 3D experiment) from spectrometer
Camel has nice features for scheduling instrument time
Camel provides interface for archiving expression plasmids, cleavage sites, etc.
automatic analysis software
Robert Kaptein "Validation of Biomolecular NMR Structures"
Tools for validation of structures. ~97 structures
Aqua available with constraints in 1996. Analysis done for
What If these 97. Now ~1200
Violating - these can be manipulated (by editing)
rms viol. - should be less than ~0.1Å
Many NMR structures do not have good Ramachandran plots.
-bond lengths - OK - problems mainly due to access S-H
-bond angles - OK
-Ramachandran plots - bad constraints - problems in XPLOR - due to scaling vdw radius by 0.8
-planarity - peptide XPLOR, DIANA - too restrictive
side chain planarity XPLOR is problematic. too loose
deviations also seen in DIANA
Nomenclature - Prochiral methylene and methyl groups often supplied.
= observed NOEs x 100 %
What is "expected" NOE. HN, Ha, Hb2, Hb3, then 1 H-atom per methylene pair
can expect to see ~60% completeness even in disordered regions - defined specifically for each amino acid type
-eliminate "redundant" constraints
-do no average coordinate
-report conditions for all protons consistent with protination state
-proper balance between experimental and energetic constraints
interface to NMRView, Xeasy, Aurelia, CNS using python in the interface
to CNS 30 min/structure on R10000 processor
PECKAY: Protein Structure Elucidation by Iteration Calculation using Ambiguous NOEs. NOESY spectra -> XPLOR input file. Iterative constraints
Rescue: available http://www.infobiogud.univ-montpl.fr Neural networks for spin system typing
Domaille: ? repeat Nature ~Oct 1997 p999 NMR
Nature ~Fall 1998 Mol. Replacement in Complex
wuld provide all data - NOESY folds different