Crystallogaphy -- lecture 25. Protein guided tours: the meaning of “ Life ”. All life is based on reduced carbon. anoxic. oxidizing. 4*10 9 y. 3*10 9. 2*10 9. 1*10 9. present. origin of life. Chloroplasts. origin of oxygenic phototrophs . oxygenated environment.
Protein guided tours: the meaning of “Life”
origin of life
origin of oxygenic phototrophs
Thanks to Doug Whittet, RPI Physics
A narrowly avoided catastrophe for life on earth:
chloroplasts are descendents of the early cyanobacteria.
Download and display 1JB0.pdb (one jay bee zero)
restrict !protein and !hohcolor cpkDisplay -> ball and stickselect magnesiumlabel %rset fontsize 12set fontstroke 2color labels yellow
Find the pseudo 2-fold axis
How many Mg are there?
What are the residue numbers of the “special pair” of chlorophylls?
(select the special pair using select XXX or YYY)spacefillselect hetero and !hohlabels offcolor temperature
How are the B-factorsdistributed?
Was NCS 2-fold symmetry enforced during refinement?
Which side is more ordered? Chain A or chain B?
Guess what: 2-fold symmetry was not enforced during evolution!
Find the name of the lipid that does not havea phosphate group.
Unix shortcut: use grepgrep ^”HETNAM” 1JB0.pdb
Characterize the environmentof the lipid. Could it have a role in the light harvest process?
select [LMG]restrict selectedcenter selectedselect within (11., [LMG]) and proteinDisplay -> ball_and_stickcolor cpkselect within (11., [LMG]) and ligand
select within (11., [LMG]) and ligandspacefill 1.5color green select within (11., [LMG]) and *.MGspacefill 1.5color white select within (11., [LMG]) and [PQN]color red select within (11., [LMG]) and solventspacefill 1.0color cyan
What is PQN?How close is it to the nearestmagnesium
restrict ligandwireframecolor cpkDisplay-> ball and stickselect [CL1] or [CL2]wireframe color greenselect [PQN]color magentaspacefill 1.0select [BCR]color orangespacefill 1.5select *.MGspacefill 1.0color white
Light harvesting complex
Trace the path of the electronsfrom the special pair to the twoquinones.
Are are of the pigmentsconnected to the special pair?
restrict [PQN]spacefill color cpkselect within (11.,[PQN]) and proteinwireframe 0.5color cpkselect within (11.,[PQN]) and ligand and not [PQN]color greenwireframe 0.5select within (11.,[PQN]) and solvent spacefill 0.6color cyan
Environment of the quinones
Which quinone is more loosely-bound
How does the electron getfrom one quinone to theother? What protein sidechainforms a bridge?
Draw a TOPS diagram of chain D
residues 24-95 only
Look in scop.berkeley.edu for the
fold under alpha+betaWhat fold is this?
RUBISCO = Ribulose Bisphophate Carboxylase/Oxygenase
---> sugars, etc.
5 + 1 = 3 + 3
One of the substrates is small and feature-less. Gets confused with O2.
Unwanted competing reaction
Download 8RUC.pdb from www.rcsb.org
color --> chain
set unitcell on
8RUC space group is C2221
What crystal class is this?
edit 8RUC.pdb (vi or jot)
Note resolution of the data. Number of reflections. Number of atoms. Number of solvent atoms.
Find “HETNAM” records.
These are the “hetero” groups.
Find the CAP and KCX “ATOM” records.
Check the B-factors. How well ordered are they?
Adjust the window to eye separation. Relax eyes to see stereo.
Trace the chain from N to C.
How many domains are there?
What “fold” is each domain? Find it in SCOP and/or CATH.
restrict within (10., 201:A)
center within (10., 201:A)
select 201:A | CAP:A
select hetero & within (10., 201:A) & MG
select hetero & within (10., 201:A) & HOH
Which residues are coordinating the magnesium?
Where is the CO2 that was added to the ribulose?
select :A & not within (10., 201:A)
color-->group (this colors from N to C)
Where is the active site relative to the domain?
select not :A & protein
color --> chain
Where is the active site relative to the oligomeric contacts?
select not :A & within (10., :A)
What chains are in contact with the A chain?
What residues are involved?
What command would select only waters that arewithin 5A of both chain A and chain L?
Like CH2, NH3 was plentiful before oxygenic phototrophs. Since then, most N in the atmosphere is in the form of N2.
Nitrogenase converts N2 to NH3
The Fe-protein cluster passes e- to the P-cluster, which passes them to the FeMo-cluster. e- accumulate at the FeMo-cluster, where the reduction of N2 takes place.
A protein conformational change might favor oxidized over reduced, driving the reaction forward.
from Rees & Howard (2000) Current Opinion in Chemical Biology, 4(5):p559-566.
dissociation of complex is rate-limiting
less than 6 e-?
N2 + 6H+
This reaction won’t happen until there are an accumulated 6e–
Conformational changes couple hydrolysis of ATP with oxidation potential of Fe in Fe-protein
The stoichiometry of nitrogenase is still not completely known.
N2 + (6+2n)H++ (6+2n)e– + p(6+2n)ATP-->
2NH3 + nH2 + p(6+2n)ADP + p(6+2n)Pi
n=number of H2 molecules formed (1 or 2, unknown)
p=number of ATP required per electron (probably 2)
O2 (molecular oxygen) inactivates
CO (carbon monoxide) inhibits (competitive)
HCCH (acetylene) substrate
HCN (cyanide) substrate
N3- (azide) substrate
N2 binds with KM = 0.02 atm
His 195A --> Glu Blocks N-fixation but allows reduction of acetylene.
Gly 69A --> Ser Blocks reduction of acetylene, but allows N-fixation.
Download 1N2C.pdb from www.rcsb.org
In RasMol answer the following questions using the scripts provided plus any additions of your own:
Display-->backbonecolor-->chainselect 50 and alphalabel %cset fontsize 20set fontstroke 4color labels yellow
line up the molecule along the non-crystallographic 2-fold.
There are 8 chains in the asu. Which chains are related to which by non-crystallographic symmetry?
select 68-69:Awireframe 50restrict selectedcenter selectedcolor cpk
Line up atoms 69:A n and 69:A ca to measure the phi angle. R-handed is positive.
Does Gly69A have a positive phi-angle?
Mutating G69 blocks reduction of acetylene, but allows N2-fixation. Would mutating Gly69A to a Serine possibly change its conformation?
Draw a TOPS diagram of chain E.
restrict :Ecenter selectedDisplay-->cartooncolor-->structureLine the structure up with the beta sheet perpendicular to the screen. Ignore short helices (they are not really helices). Draw strands as up or down arrows and the helices as circles. Then draw connecting lines, to the middle if the connection is toward you, to the edge if the connection is away from you. Find the N-term. Number the strands from N to C. Find the fold class from SCOP (scop.berkeley.edu). (goto “top of the heirarchy” then class 3, “alpha and beta proteins”)
Chain E is “3-layer”. When you have numbered the strands, look in SCOP for the “Fold” with the observed strand order (for example: 43125, etc). (cute trick: use the browser’s “search in page” function).
The strand order can be read from right-to-left or left-to-right. If a terminal strand is at the edge of the beta sheet it might be missing. Also, extra strands might be added at the C-term or N-term is it occurs at the sheet edge.
Write the fold name here ______________________
Characterize the environment of the ADP and metal clusters.
select within (6., hetero) and (:A | :B | :E | :F)restrict selectedDisplay -->ball and stickcolor whiteselect selected and heterospacefillcolor cpkselect within (6., hetero) and (:A | :B | :E | :F)select selected and acidiccolor red select within (6., hetero) and (:A | :B | :E | :F)select selected and basiccolor blue
Characterize the environment of the ADP and metal clusters.
select within (6., hetero) and (:A | :B | :E | :F)select selected and polar and not basic and not acidiccolor green
How would you characterize the binding sites? (check one for each het group)
mostly mostly mostly mixed non- basic acidic polar charges polarADPFS4CLFCFMCA